Methods of production of products of metabolic pathways

ABSTRACT

A plurality of isolated polynucleotide sequences encoding enzymes of the astaxanthin pathway is disclosed. The polynucleotides include:
         (i) a polynucleotide which encodes Phytoene dehydrogenase (crtI) and a first transcriptional regulatory sequence;   (ii) a polynucleotide which encodes Beta-lycopene cyclase (lcy-B) and a second transcriptional regulatory sequence;   (iii) a polynucleotide which encodes Beta-carotene ketolase (crtW) and a third transcriptional regulatory sequence; and   wherein the first, second and third regulatory sequence are selected such that the expression of the Icy-B and the crtW is greater than a level of expression of the crtI. Methods of generating astaxanthin using the plurality of polynucleotide are also disclosed as well as bacterial cells comprising high levels of astaxanthin.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.14/415,612 filed on Jan. 19, 2015, which is a National Phase of PCTPatent Application No. PCT/IL2013/050606 having International FilingDate of Jul. 17, 2013, which claims the benefit of priority of U.S.Provisional Patent Application No. 61/672,796 filed on Jul. 18, 2012.The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 70214SequenceListing.txt, created on Jun. 2,2017, comprising 45,281 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates tobiotechnological methods for generation of products of metabolicpathways such as astaxanthin.

Native protein abundance in bacteria spans over five orders ofmagnitude. The balancing of protein expression levels is at the heart ofproper functioning of natural biological systems and is often criticalto metabolic engineering efforts. While the manipulation ofintracellular protein levels, such as strong over and under-expressionis widely used to elucidate the functions of biological systems, anability to fine-tune the levels of many genes in parallel is a majoroutstanding challenge. In contrast to native biological systems, wherethe balancing of protein levels is selected for during evolution, theexpression of a synthetic system can lead to imbalances in proteinconcentrations. As a result, synthetic pathways rarely functionoptimally when first introduced and the enzyme levels must befine-tuned.

FIG. 15A schematically depicts the major challenges associated withimbalanced enzyme concentrations based on a two-step metabolic pathwaymodel. First, low enzyme expression can limit the pathway flux andtherefore product synthesis rate (blue region). At the other extreme,excessive expression might lead to protein burden, resulting in thedepletion of cellular resources that limit growth (purple region).Finally, imbalances between enzymes producing and consuming anintermediate metabolite can result in a metabolic bottleneck and a highconcentration of potentially toxic pathway intermediates (green region).

Approaches for controlling the intracellular abundance of proteinsinclude altering the promoter K. Hammer, I. Mijakovic, P. R. Jensen,Synthetic promoter libraries—tuning of gene expression, Trends inBiotechnology 24, 53-55 (2006)] or the ribosome binding site (RBS) [H.M. Salis, E. A. Mirsky, C. A. Voigt, Automated design of syntheticribosome binding sites to control protein expression, Nat Biotechnol 27,946-950 (2009); H. H. Wang et al., Programming cells by multiplex genomeengineering and accelerated evolution, Nature 460, 894-898 (2009)]sequences, modulating the stability of transcripts and varying thedegradation rate of the mature protein.

Carotenoids, such as astaxanthin, are natural pigments that areresponsible for many of the yellow, orange and red colors seen in livingorganisms. Carotenoids are widely distributed in nature and have, invarious living systems, two main biological functions: they serve aslight-harvesting pigments in photosynthesis, and they protect againstphoto oxidative damage.

Astaxanthin is the most expensive commercially used carotenoid compound(today's market value is greater than 3,500 $/kg). It is utilized mainlyas nutritional supplement which provides pigmentation in a wide varietyof aquatic animals. In the Far-East it is used also for feeding poultryto yield a typical pigmentation of chickens. It is also a desirable andeffective nontoxic coloring for the food industry and is valuable incosmetics. Recently it was reported that astaxanthin is a potentantioxidant in humans and thus is a desirable food additive.

Although astaxanthin is synthesized in a variety of bacteria, fungi andalgae, the key limitation to the use of biological systems for itsproduction is its low yield. One of the reasons for the low yield is thecomplexity of the astaxanthin pathway, whereby 7 genes of the pathwaymust be expressed in the cells for efficient expression. Fine tuning ofthe amount of expression of each of these genes is essential foroptimizing astaxanthin expression.

Lemuth et al., [Microbial Cell Factories 10, 29, 2011] teachesexpression of astaxanthin in E. coli.

Salis et al., Nature Biotechnology Volume 27, No. 10, pages 946-950,2009 teaches sequences of ribosome binding sites.

U.S. Patent Application No. 20120015849 teaches a method of creating DNAlibraries that include an artificial promoter library and/or a modifiedribosome binding site library and transforming bacterial host cells withthe library to obtain a population of bacterial clones having a range ofexpression levels for a chromosomal gene of interest.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a plurality of isolated polynucleotide sequencesencoding enzymes of the astaxanthin pathway comprising:

(i) a polynucleotide which encodes Phytoene dehydrogenase (crtI) and afirst transcriptional regulatory sequence;

(ii) a polynucleotide which encodes Beta-lycopene cyclase (lcy-B) and asecond transcriptional regulatory sequence;

(iii) a polynucleotide which encodes Beta-carotene ketolase (crtW) and athird transcriptional regulatory sequence; and

wherein the first, second and third regulatory sequence are selectedsuch that the expression of the Icy-B and the crtW is greater than alevel of expression of the crtI.

According to an aspect of some embodiments of the present inventionthere is provided a bacterial cell comprising more than 2 mg/g cell dryweight of astaxanthin.

According to an aspect of some embodiments of the present inventionthere is provided a bacterial cell comprising more than 5 mg/g cell dryweight of astaxanthin.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating astaxanthin comprisingexpressing polynucleotides encoding enzymes of an astaxanthin pathway,the polynucleotides comprising:

(i) a polynucleotide which encodes Phytoene dehydrogenase (crtI) and afirst transcriptional regulatory sequence;

(ii) a polynucleotide which encodes Beta-lycopene cyclase (lcy-B) and asecond transcriptional regulatory sequence;

(iii) a polynucleotide which encodes Beta-carotene ketolase (crtW) and athird transcriptional regulatory sequence; and

wherein the first, second and third regulatory sequence are selectedsuch that the expression of the lcy-B and the crtW is greater than alevel of expression of the crtI.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating astaxanthin comprisingexpressing polynucleotides encoding enzymes of an astaxanthin pathway,the polynucleotides comprising:

(i) a polynucleotide which encodes Isopentenyl pyrophosphate (idi) and afirst transcriptional regulatory sequence;

(ii) a polynucleotide which encodes Geranylgeranyl pyrophosphatesynthase (crtE) and a second transcriptional regulatory sequence;

(iii) a polynucleotide which encodes Prephytoene pyrophosphate synthase(crtB) and a third transcriptional regulatory sequence;

(iv) a polynucleotide which encodes Phytoene dehydrogenase (crtI) and afourth transcriptional regulatory sequence;

(v) a polynucleotide which encodes Beta-lycopene cyclase (lcy-B) and afifth transcriptional regulatory sequence;

(vi) a polynucleotide which encodes Beta-carotene ketolase (crtW) and asixth transcriptional regulatory sequence; and

(vii) a polynucleotide which encodes Beta-carotene hydroxylase (crtZ)and a seventh transcriptional regulatory sequence

wherein the fourth, fifth and sixth regulatory sequence are selectedsuch that the expression of the lcy-B and the crtW is greater than alevel of expression of the crtI.

According to an aspect of some embodiments of the present inventionthere is provided a plurality of isolated polynucleotide sequencesencoding enzymes of the astaxanthin pathway comprising:

(i) a polynucleotide which encodes Isopentenyl pyrophosphate (idi) and afirst transcriptional regulatory sequence;

(ii) a polynucleotide which encodes Geranylgeranyl pyrophosphatesynthase (crtE) and a second transcriptional regulatory sequence;

(iii) a polynucleotide which encodes Prephytoene pyrophosphate synthase(crtB) and a third transcriptional regulatory sequence;

(iv) a polynucleotide which encodes Phytoene dehydrogenase (crtI) and afourth transcriptional regulatory sequence;

(v) a polynucleotide which encodes Beta-lycopene cyclase (lcy-B) and afifth transcriptional regulatory sequence;

(vi) a polynucleotide which encodes Beta-carotene ketolase (crtW) and asixth transcriptional regulatory sequence; and

(vii) a polynucleotide which encodes Beta-carotene hydroxylase (crtZ)and a seventh transcriptional regulatory sequence

wherein the fourth, fifth and sixth regulatory sequence are selectedsuch that the expression of the Icy-B and the crtW is greater than alevel of expression of the crtI.

According to some embodiments of the invention, the plurality ofisolated polynucleotide sequences further comprises at least one of:

(iv) a polynucleotide which encodes Isopentenyl pyrophosphate (idi) anda fourth transcriptional regulatory sequence; or

(v) a polynucleotide which encodes Geranylgeranyl pyrophosphate synthase(crtE) and a fifth transcriptional regulatory sequence; or

(vi) a polynucleotide which encodes Prephytoene pyrophosphate synthase(crtB) and a sixth transcriptional regulatory sequence; or

(vii) a polynucleotide which encodes Beta-carotene hydroxylase (crtZ)and a seventh transcriptional regulatory sequence.

According to some embodiments of the invention, each of the firstregulatory sequence, said second regulatory sequence and said thirdregulatory sequence is a ribosome binding site (RBS).

According to some embodiments of the invention, each of the firstregulatory sequence, the second regulatory sequence and the thirdregulatory sequence is a promoter.

According to some embodiments of the invention, the first regulatorysequence, the second regulatory sequence and the third regulatorysequence are selected such that the expression of said lcy-B and saidcrtW is at least ten times greater than a level of expression of saidcrtI.

According to some embodiments of the invention, a sequence of an RBS of(i) is selected to bring about expression of the crtI to at least 80% ofthe extent as the RBS having a sequence as set forth in SEQ ID NO: 4, 5,6 or 7;

wherein a sequence of an RBS of (ii) is selected to bring aboutexpression of the lcy-B to at least 80% of the extent as the RBS havinga sequence as set forth in SEQ ID NO: 2 or 3;

wherein a sequence of an RBS of (iii) is selected to bring aboutexpression of the crtW to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 2 or 3.

According to some embodiments of the invention, a sequence of an RBS of(iv) is selected to bring about expression of the crtI to at least 80%of the extent as the RBS having a sequence as set forth in SEQ ID NO: 4,5, 6 or 7;

wherein a sequence of an RBS of (v) is selected to bring aboutexpression of the lcy-B to at least 80% of the extent as the RBS havinga sequence as set forth in SEQ ID NO: 2 or 3;

wherein a sequence of an RBS of (vi) is selected to bring aboutexpression of the crtW to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 2 or 3.

According to some embodiments of the invention, a sequence of an RBS of(i) is selected to bring about expression of the idi to at least 80% ofthe extent as the RBS having a sequence as set forth in SEQ ID NO: 5;

wherein a sequence of an RBS of (ii) is selected to bring aboutexpression of the crtE to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 5;

wherein a sequence of an RBS of (iii) is selected to bring aboutexpression of the crtB to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 3;

wherein a sequence of an RBS of (iv) is selected to bring aboutexpression of the crtI to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 7;

wherein a sequence of an RBS of (v) is selected to bring aboutexpression of the lcy-B to at least 80% of the extent as the RBS havinga sequence as set forth in SEQ ID NO: 2;

wherein a sequence of an RBS of (v) is selected to bring aboutexpression of the crtW to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 3; and

wherein a sequence of an RBS of (vi) is selected to bring aboutexpression of the crtZ to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 6.

According to some embodiments of the invention, a sequence of an RBS of(i) is selected to bring about expression of the idi to at least 80% ofthe extent as the RBS having a sequence as set forth in SEQ ID NO: 6;

wherein a sequence of an RBS of (ii) is selected to bring aboutexpression of the crtE to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 3;

wherein a sequence of an RBS of (iii) is selected to bring aboutexpression of the crtB to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 7;

wherein a sequence of an RBS of (iv) is selected to bring aboutexpression of the crtI to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 5;

wherein a sequence of an RBS of (v) is selected to bring aboutexpression of the lcy-B to at least 80% of the extent as the RBS havinga sequence as set forth in SEQ ID NO: 2;

wherein a sequence of an RBS of (v) is selected to bring aboutexpression of the crtW to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 2; and

wherein a sequence of an RBS of (vi) is selected to bring aboutexpression of the crtZ to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 2.

According to some embodiments of the invention, the plurality ofpolynucleotide sequences further comprise:

(viii) a polynucleotide encoding a deoxyxylulose-5-phosphate synthase(DXS) and an eighth transcriptional regulatory sequence.

According to some embodiments of the invention, the eighthtranscriptional regulatory sequence is selected to bring aboutexpression of the DXS to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 6.

According to some embodiments of the invention, the plurality ofpolynucleotide sequences are comprised in a single expression vector.

According to some embodiments of the invention, the plurality ofpolynucleotide sequences are comprised in a plurality of expressionvectors.

According to some embodiments of the invention, each of the RBS isflanked by a spacer sequence.

According to some embodiments of the invention, the spacer sequenceupstream of each of the RBS is at least 80% homologous to the sequenceas set forth in SEQ ID NO: 8.

According to some embodiments of the invention, the spacer downstream ofeach of the RBS is at least 80% homologous to the sequence as set forthin SEQ ID NO: 9.

According to some embodiments of the invention, the bacterial cell is anE. coli cell.

According to some embodiments of the invention, the astaxanthin isexpressed in an inclusion body in the bacterial cell.

According to some embodiments of the invention, the bacterial cell isgenetically modified.

According to some embodiments of the invention, the bacterial cellexpresses the plurality of polynucleotides of the present invention.According to some embodiments of the invention, each of the firstregulatory sequence, the second regulatory sequence, the thirdregulatory sequence, the fourth regulatory sequence, the fifthregulatory sequence, the sixth regulatory sequence and the seventhregulatory sequence is a RBS.

According to some embodiments of the invention, the regulatory sequencesare selected such that the expression of the lcy-B and the crtW is atleast ten times greater than a level of expression of the crtI.

According to some embodiments of the invention, the sequence of the RBSof (iv) is selected to bring about expression of the crtI to at least80% of the extent as the RBS having a sequence as set forth in SEQ IDNO: 4, 5, 6 or 7;

wherein a sequence of the RBS of (v) is selected to bring aboutexpression of the lcy-B to at least 80% of the extent as the RBS havinga sequence as set forth in SEQ ID NO: 2 or 3;

wherein a sequence of the RBS of (vi) is selected to bring aboutexpression of the crtW to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 2 or 3.

According to some embodiments of the invention, the sequence of the RBSof (i) is selected to bring about expression of the idi to at least 80%of the extent as the RBS having a sequence as set forth in SEQ ID NO: 5;

wherein a sequence of the RBS of (ii) is selected to bring aboutexpression of the crtE to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 5;

wherein a sequence of the RBS of (iii) is selected to bring aboutexpression of the crtB to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 3;

wherein a sequence of the RBS of (iv) is selected to bring aboutexpression of the crtI to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 7;

wherein a sequence of the RBS of (v) is selected to bring aboutexpression of the lcy-B to at least 80% of the extent as the RBS havinga sequence as set forth in SEQ ID NO: 2;

wherein a sequence of the RBS of (vi) is selected to bring aboutexpression of the crtW to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 3; and

wherein a sequence of the RBS of (vii) is selected to bring aboutexpression of the crtZ to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 6.

According to some embodiments of the invention, the sequence of the RBSof (i) is selected to bring about expression of the idi to at least 80%of the extent as the RBS having a sequence as set forth in SEQ ID NO: 6;

wherein a sequence of the RBS of (ii) is selected to bring aboutexpression of the crtE to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 3;

wherein a sequence of the RBS of (iii) is selected to bring aboutexpression of the crtB to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 7;

wherein a sequence of the RBS of (iv) is selected to bring aboutexpression of the crtI to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 5;

wherein a sequence of the RBS of (v) is selected to bring aboutexpression of the lcy-B to at least 80% of the extent as the RBS havinga sequence as set forth in SEQ ID NO: 2;

wherein a sequence of the RBS of (vi) is selected to bring aboutexpression of the crtW to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 2; and

wherein a sequence of the RBS of (vii) is selected to bring aboutexpression of the crtZ to at least 80% of the extent as the RBS having asequence as set forth in SEQ ID NO: 2.

According to some embodiments of the invention, the method furthercomprises introducing into the cell a polynucleotide encoding adeoxyxylulose-5-phosphate synthase (DXS).

According to some embodiments of the invention, a sequence of an RBS ofthe polynucleotide expressing DXS is selected to bring about expressionof the DXS to at least 80% of the extent as the RBS having a sequence asset forth in SEQ ID NO: 6.

According to some embodiments of the invention, the expressing iseffected in a bacterial cell.

According to some embodiments of the invention, the bacterial cellcomprises an E. coli cell.

According to some embodiments of the invention, each of the RBS isflanked by a spacer sequence.

According to some embodiments of the invention, the spacer sequenceupstream of each of the RBS is at least 80% homologous to the sequenceas set forth in SEQ ID NO: 8.

According to some embodiments of the invention, the spacer downstream ofeach of the RBS is at least 80% homologous to the sequence as set forthin SEQ ID NO: 9.

According to some embodiments of the invention, each of thepolynucleotides are comprised on a single expression vector.

According to some embodiments of the invention, the method furthercomprises isolating the astaxanthin following the expressing.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide comprising:

(i) a first RBS operatively linked to a first enzyme coding sequence;

(ii) a second RBS operatively linked to a second enzyme coding sequence;and

(iii) a third RBS operatively linked to a third enzyme coding sequence;

wherein the second RBS is selected such that the level of expression ofthe second enzyme coding sequence is greater than the level ofexpression of the first enzyme coding sequence;

wherein the third RBS is selected such that the level of expression ofthe third enzyme coding sequence is greater than the level of expressionof the second enzyme coding sequence;

wherein the first enzyme, the second enzyme and the third enzyme arenon-identical enzymes and each part of a biosynthesis pathway of anidentical product of interest.

According to some embodiments of the invention, the isolatedpolynucleotide further comprises:

(iv) a fourth RBS operatively linked to a fourth enzyme coding sequence.

According to some embodiments of the invention, the fourth enzyme isnon-identical to the first, second and third enzyme and is part of abiosynthesis pathway of the identical product.

According to some embodiments of the invention, the isolatedpolynucleotide further comprises:

(v) a fifth RBS operatively linked to a fifth enzyme coding sequence.

According to some embodiments of the invention, the fifth enzyme isnon-identical to the first, second, third and fourth enzyme and is partof a biosynthesis pathway of the identical product of interest.

According to some embodiments of the invention, the isolatedpolynucleotide further comprises:

(vi) a sixth RBS operatively linked to a sixth enzyme coding sequence.

According to some embodiments of the invention, the sixth enzyme isnon-identical to the first, second, third, fourth and fifth enzyme andis part of a biosynthesis pathway of the identical product of interest.

According to some embodiments of the invention, each of the RBS isflanked by a spacer sequence.

According to some embodiments of the invention, the spacer sequenceupstream of each of the RBS is at least 80% homologous to the sequenceas set forth in SEQ ID NO: 8.

According to some embodiments of the invention, the spacer downstream ofeach of the RBS is at least 80% homologous to the sequence as set forthin SEQ ID NO: 9.

According to some embodiments of the invention, the product of interestis a protein.

According to some embodiments of the invention, the product of interestis selected from the group consisting of a food product, apharmaceutical and a fuel.

According to an aspect of some embodiments of the present inventionthere is provided a method of selecting polynucleotide sequences forsynthesizing an optimal amount of a product of interest which is theproduct of a biosynthesis pathway comprising at least three enzymes, themethod comprising:

(a) introducing the polynucleotide described herein into a cell underconditions which allow synthesis of the product of interest in the cell;and

(b) measuring the amount of the product of interest, wherein an amountof the product of interest is indicative of the polynucleotide sequencesto be selected.

According to some embodiments of the invention, the cell is selectedfrom the group consisting of a bacterial cell, a mammalian cell, a plantcell, a fungal cell and an algae cell.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 illustrates a modular cloning strategy for combinatorial assemblyof multi-gene constructs. A method of eliminating false-positive clonesby a direct selection for correctly assembled constructs was developed.Each gene of interest was joined with a chloramphenicol (Cm) resistancecassette flanked by NheI (‘N’) and PciI (‘P’) restriction sites. Thesequence designated for assembly contains an upstream SpeI (‘S’)restriction site. To assemble the first target sequence into a vectorthe DNA was first digested using SpeI and PciI, followed by ligation andtransformation. Cells were plated on a selective agar supplemented withCm. Since the backbone vector does not include Cm resistance, onlyclones which were properly assembled (i.e. contain the designatedsequence and the resistance marker) will be able to form colonies. Toincorporate the next target sequence, the assembly product was extractedfrom the cells and digested with NheI and PciI, effectively discardingthe resistance marker from the construct. The second target sequence, asthe first one, was digested using SpeI and PciI and assembled to thevector. Importantly, the sticky ends of NheI and SpeI restriction sitesare compatible and joined together to form a scar, a sequence thatcannot be cleavage by either NheI or SpeI (‘x’). After the secondassembly round the new construct now contains both target sequences andthe Cm resistance, enabling once more a direct selection for positiveconstructs. This sequence of events can be repeated to assemblemulti-gene operons.

FIG. 2 illustrates RBS modulation of a target ORF. The desired codingsequence is paired with the resistance cassette and then assembled upona vector containing a RBS upstream to the insertion site. Barcoded andnon-barcoded assemblies rely on the same logic but differ in therestriction sites due to technical reasons.

FIG. 3 illustrates the pNiv backbone plasmid. RRNB terminator was placedupstream to the multiple cloning site to minimize leaky expressionthroughout the assembly process.

FIG. 4 is a schematic representation of the insulated RBS unit design.The composition of the flanking sequences has been shown to affect theexpression level of a given RBS. In an effort to minimize such secondaryeffects, a constant spacer and tag sequences were introduced up- anddownstream to the RBS sequence.

FIG. 5 illustrates the pSB4K5:Ptac—the expression plasmid. The hybridPtac promoter was placed on a pSB4K5 plasmid backbone, upstream to themultiple cloning site.

FIGS. 6A-6D are graphs illustrating flow cytometry fluorescencemeasurements of the RBS set using different fluorescence proteins andinduction conditions.

FIG. 7 is a graph comparing predicted RBS activity to experimentalfluorescence measurements. CFP (blue), YFP (green) and mCherry (red)reporters were each paired to an RBS sequences (A-F, as denoted byletters). The x-axis represents the predicted RBS activity for eachsequence while the y-axis represents the measured fluorescence levels.Fluorescence levels were normalized so the mid-point of the predicteddynamic range—(Predicted_(max)-Predicted_(min)/2)—corresponds to themid-point of the experimentally measured dynamic range for eachreporter—(Measured_(max)−Measured_(min)/2).

FIG. 8 illustrates single tube combinatorial assembly. An equi-molarmixture of pNiv:RBS plasmids with six distinct RBS sequence was used inthe assembly reaction. The target coding sequence was ligated to theplasmid mix to yield six distinct products all containing the samecoding sequence but with a different RBS upstream located upstream toit.

FIG. 9 illustrates the assembly of a synthetic operon of RBS modulatedgenes.

FIG. 10 illustrates assembly of a synthetic operon with barcoded RBSsites. At each assembly step the barcode corresponding to the newlyassembled RBS modulated coding sequence is stacked at the 3′ end of theoperon.

FIG. 11 illustrates single tube combinatorial assembly of barcoded RBSmixture. The target insert is ligated with pRBS-barcoded mixture, theresulting library contains the target coding sequence with different RBSsequence upstream to it.

FIG. 12 is a tricolor reporter operon—plasmid map.

FIG. 13 is a graph illustrating the dependence of the second gene in theoperon (mCherry) on first gene (YFP). The RBS sequence controllingmCherry is denoted using colors (blue for RBS A, red for RBS C, andgreen for RBS E), and RBS sequences controlling YFP corresponds toshapes (asterisk for RBS A, circle for RBS B, triangle for RBS C, squarefor RBS D, cross for RBS E, and stars for RBS F). The effect oftranslational coupling is evident, where the expression level of YFPmodulates the expression of mCherry. The dependency between YFP andmCherry levels follows a linear trend in log space with a slope of ˜⅓.

FIG. 14 is a carotenoids biosynthesis operon—plasmid map.

FIGS. 15A-15D illustrates that modulation of enzyme expression levels isrequired for balanced pathway function. (A) A simple quantitative modelbased on a reversible Michaelis-Menten kinetics that consists of twoenzymes was used to depict the outcomes of an unbalanced enzymeexpression. Only a small region of the enzyme concentrations space,shown in white, sustains optimal production. (B) A small set of RBSsequences spans several orders of magnitudes of protein expression. Sixpre-characterized RBS sequences were employed and paired to genes ofinterest downstream to an inducible promoter.

(SEQ ID NO: 2) #8 (RBS-A): AGGAGGTTTGGA (SEQ ID NO: 3) #1 (RBS-B):AACAAAATGAGGAGGTACTGAG (SEQ ID NO: 4) #17 (RBS-C): AAGTTAAGAGGCAAGA(SEQ ID NO: 5) #27 (RBS-D): TTCGCAGGGGGAAG (SEQ ID NO: 6) #20 (RBS-E):TAAGCAGGACCGGCGGCG (SEQ ID NO: 7) “Dead-RBS” (RBS-F) : CACCATACACTG 

(C) Flow cytometry fluorescence measurement of YFP paired with the setof RBS sequences (15). (D) A modular cloning strategy for combinatorialassembly of multi-gene constructs which enables the barcoding ofassembled parts and direct selection for correctly assembled constructs.Each gene of interest is joined with a chloramphenicol (Cm) resistancecassette and paired with a library of RBS sequences. Once the first geneis assembled into the vector, properly assembled clones are selected forCm resistance. To insert the next gene, the marker is discarded and theadditional part is assembled into the vector. The newly formed constructcontains the two RBS-modified genes and a resistance marker, enablingonce more a direct selection for positive constructs. This sequence ofsteps can be repeated to easily assemble a combinatorial library ofRBS-modulated multi-gene operons.

FIGS. 16A-16D illustrate that RBS modulation of three fluorescentproteins spans a color space. (A) CFP, YFP and mCherry werecombinatorially joined with three representatives of the present RBS set(sequences ‘A’, ‘C’ and ‘E’), and the genes were assembled together. Theresulting operon library differs only in the RBS sequences regulatinggene expression. (B) A fluorescence microscopy imaging of E. colicolonies, transformed with an operon library. The observed colorsrepresent additive combinations of the three primary colors, assigned toeach of the fluorescent proteins. Irregular colony shapes are the resultof touching boundaries of adjacent colonies. Some colonies harboringweak RBS appear black. Inset: a brightfield image. (C) Fluorescenceimaging of E. coli colonies containing the tricolor RBS modulatedoperon. The images are arranged on a 3D grid where the position on eachaxis corresponds to the RBS strength of the fluorescent proteins. (D)YFP and mCherry fluorescence levels of clones sampled from a two-coloroperon library. RBS composition, as determined by barcode sequencing(see supporting online text) is shown. Identical genotypes (each labeledin a distinct color) cluster together in the fluorescence space. Theeffect of translational coupling is also evident, where the expressionlevel of YFP modulates the expression of mCherry by up to half an orderof magnitude.

FIGS. 17A-1C illustrate that carotenoid accumulation profile varies withthe RBS sequences of biosynthetic genes. (A) A library of syntheticoperons differing in the RBS sequences regulating each of the sevengenes of the carotenoid biosynthesis pathway was generated. (B) Abinocular microscopy imaging of E. coli colonies transformed with theoperon library. The color of the colony corresponds to the compositionof the accumulated carotenoids, each having a characteristic color. (C)The carotenoid accumulation profile and RBS composition of clonessampled from the transformed library. The RBS composition of eachsampled clone was determined by sequencing (RBS encoding in barcoderefers to the order of genes as illustrated in 3A) and the carotenoidprofile of each clone was analyzed using HPLC. Different genotypesresult in distinct phenotypes, i.e. distinct carotenoids accumulationprofiles. Circle area indicates the production yield of a carotenoidintermediate, according to the metabolic pathway described on the right(15).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to abiotechnological method for generation of products of metabolic pathwayssuch as astaxanthin.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Tuning the expression of recombinant enzymes is essential for theoptimization of a metabolic pathway (FIG. 15A). There are two mainstrategies to achieve balanced expression levels. Rational designinvolves the calculation or estimation of the relative and absoluteamount of each of the pathway's component. However, such attempts areoften limited by the lack of sufficient information regarding thekinetics, energetics, and regulation of pathway components.Alternatively, strategies based on random mutagenesis of regulatoryelements can sample the expression space and screen or select for adesired phenotype. Yet, even a large pool of mutants does not ensureadequate coverage of the expression space: often, the vast majority ofgenotypes are clustered in a small portion of the phenotypic space.Moreover, random mutagenesis often yields large libraries in whichscreening for a desired phenotype can be challenging or even infeasible.

The present inventors introduced a strategy that facilitates theexploration of the phenotypic space using a compact set of regulatoryelements. By employing a small set of well-characterized RBS sequencesto regulate the expression of multiple genes in a synthetic operon, thepresent inventors were able to efficiently sample the multi-dimensionalexpression space across several orders of magnitude in each axis.Importantly, the small size of the RBS set limits the number of geneticvariants in the library and enables a fast screening.

By modulating the ribosome binding sites of genes involved in carotenoidbiosynthesis, the present inventors demonstrate that the accumulation ofmetabolic products of a pathway varies significantly according to theRBS sequences regulating its constituent enzymes. The present inventorsfound that the combinatorial assembly of the astaxanthin biosyntheticpathway resulted in a 4-fold yield increase over conventional assemblyand selection methods, thereby exemplifying the strength of sampling ofthe expression space using a small set of characterized regulatoryelements.

The strategy presented herein can be expanded in various ways.Specifically, other regulatory elements can further modulate geneexpression. For example, by employing a small library of promoters tocontrol the transcription of the operon, the span of the overallexpression space can be further increased by several orders ofmagnitudes.

In order to determine the ratios of enzymes of the metabolic pathwayrequired for optimal production of the product of the metabolic pathway,the present inventors have generated a polynucleotide construct whichcomprises at least three different RBS sequences, each RBS being of adifferent strength. Introduction of the sequences encoding the relevantenzymes of the metabolic pathway into this construct in variouspermutations allows for the screening of the optimal RBS-enzymecombinations.

Thus, according to one aspect of the present invention there is providedan isolated polynucleotide comprising:

(i) a first RBS operatively linked to a first enzyme coding sequence;

(ii) a second RBS operatively linked to a second enzyme coding sequence;and

(iii) a third RBS operatively linked to a third enzyme coding sequence;

wherein the second RBS is selected such that the level of expression ofthe second enzyme is greater than the level of expression of the firstenzyme;

wherein the third RBS is selected such that the level of expression ofthe third enzyme is greater than the level of expression of the secondenzyme;

wherein the first enzyme, the second enzyme and the third enzyme arenon-identical enzymes and each part of a biosynthesis pathway of anidentical product.

The phrase “an isolated polynucleotide” refers to a single or doublestranded nucleic acid sequence which is isolated and provided in theform of an RNA sequence, a complementary polynucleotide sequence (cDNA),a genomic polynucleotide sequence and/or a composite polynucleotidesequences (e.g., a combination of the above).

As used herein the phrase “complementary polynucleotide sequence” refersto a sequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.Such a sequence can be subsequently amplified in vivo or in vitro usinga DNA dependent DNA polymerase.

As used herein the phrase “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

As used herein the phrase “composite polynucleotide sequence” refers toa sequence, which is at least partially complementary and at leastpartially genomic. A composite sequence can include some exonalsequences required to encode the polypeptide of the present invention,as well as some intronic sequences interposing therebetween. Theintronic sequences can be of any source, including of other genes, andtypically will include conserved splicing signal sequences. Suchintronic sequences may further include cis acting expression regulatoryelements.

A “ribosome binding site” (RBS) is a short nucleotide sequence usuallycomprising about 4-16 base pairs and functions by positioning theribosome on the mRNA molecule for translation of an encoded protein.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, a promoteris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Linking of nucleic acid sequences may be accomplished by ligation atconvenient restriction sites. If such sites do not exist, syntheticoligonucleotide adaptors or linkers may be used in accordance withconventional practice, such as assembly PCR.

Sequences of exemplary RBS and their relative strength may be found inSalis et al., Nature Biology, Volume 27, No. 10, 2009, page 946-950,including the supplementary information thereof, incorporated herein byreference. Preferably, the RBS is selected to one whose strength is notaffected by the downstream sequence. Methods of selecting the relativestrength of an RBS are also disclosed therein. For example, the relativestrength of an RBS may be determined by linking it to a polynucleotidesequence encoding a detectable marker and expressing the marker in acell. The marker may be a fluorescent protein, a phosphorescent proteinor any protein that may be detected using an antibody. The amount of thedetectable protein correlates with the strength of the RBS.

Exemplary RBS sequences include for example:

(SEQ ID NO: 2) (RBS-A): AGGAGGTTTGGA (SEQ ID NO: 3)(RBS-B): AACAAAATGAGGAGGTACTGAG (SEQ ID NO: 4) (RBS-C): AAGTTAAGAGGCAAGA(SEQ ID NO: 5) (RBS-D): TTCGCAGGGGGAAG (SEQ ID NO: 6)(RBS-E): TAAGCAGGACCGGCGGCG (SEQ ID NO: 7) (RBS-F): CACCATACACTG

Additional RBS sequences are as follows:

AGGAAA, (SEQ ID NO. 70), AGAAAA (SEQ ID NO. 71), AGAAGA (SEQ ID NO. 72),AGGAGA (SEQ ID NO. 73), AAGAAGGAAA (SEQ ID NO. 74), AAGGAAAA (SEQ ID NO.75), AAGGAAAG (SEQ ID NO. 76), AAGGAAAU (SEQ ID NO. 77), AAGGAAAAA (SEQID NO. 78), AAGGAAAAG (SEQ ID NO. 79), AAGGAAAAU (SEQ ID NO. 80),AAGGAAAAAA (SEQ ID NO. 81), AAGGAAAAAG (SEQ ID NO. 82), AAGGAAAAAU (SEQID NO. 83), AAGGAAAAAAA (SEQ ID NO. 84), AAGGAAAAAAG (SEQ ID NO. 85),AAGGAAAAAAU (SEQ ID NO. 86), AAGGAAAAAAAA (SEQ ID NO. 87), AAGGAAAAAAAG(SEQ ID NO. 88), AAGGAAAAAAAU (SEQ ID NO. 89), AAGGAAAAAAAAA (SEQ ID NO.90), AAGGAAAAAAAAG (SEQ ID NO. 91), AAGGAAAAAAAAU (SEQ ID NO. 92),AAGGAAAAAAAAAA (SEQ ID NO. 93), AAGGAAAAAAAAAG (SEQ ID NO. 94),AAGGAGGAAA (SEQ ID NO. 95), and AAGGAAAAAAAAAU (SEQ ID NO. 96).

According to one embodiment, the third RBS is at least 2 times, 3 times,5 times or even 10 times as strong as the second RBS.

According to another embodiment, the second RBS is at least 2 times, 3times, 5 times or even 10 times as strong as the first RBS.

It will be appreciated that depending on the number of enzymes of aparticular metabolic pathway, and depending on the number of enzymeswhich are natively expressed in the particular cell system used, thepolynucleotide may comprise additional RBSs and encode additionalenzymes. Thus, the isolated polynucleotide may comprise a fourth, afifth or a sixth RBS operatively linked to a different enzyme codingsequence which is present in the metabolic pathway.

Since the sequences flanking the transcriptional regulatory element(e.g. RBS) can affect expression levels, spacer sequences—a constantsequence of 10-50 bp, for example about 20 bp, located upstream anddownstream thereof may be inserted into the expression construct.

As used herein, the term “spacer sequence” refers to any sequence thatis either upstream or downstream of the sequence being discussed (e.g.,for genes A B C, gene B is flanked by the A and C gene sequences). Insome embodiments, a flanking sequence is present on only a single side(either 3′ or 5′) of a DNA fragment, but in preferred embodiments, it ison each side of the sequence being flanked.

An exemplary sequence that may be placed upstream of an RBS is asequence at least 70% homologous, at least 80% homologous, at least 90%homologous or 100% homologous to (SEQ ID NO: 8). An exemplary sequencethat may be placed downstream of an RBS is a sequence at least 70%homologous, at least 80% homologous, at least 90% homologous or 100%homologous to (SEQ ID NO: 9).

As mentioned herein above, the present invention further contemplatesinsertion of stabilizing mRNA sequences into the expression construct.

A “stabilizing mRNA” is a nucleic acid sequence insert used to influencegene expression. These inserts are generally located between thetranscription and translational start sites of a gene or nucleic acidsequence.

Stabilizing mRNA sequences are well known in the art and reference ismade to Carrier et al. (1999) Biotechnol. Prog. 15:58-64. Preferred mRNAstabilizing sequences include the sequences:

(SEQ ID NO. 97) GGTCGAGTTATCTCGAGTGAGATATTGTTGACG,; (SEQ ID NO. 98)GGTGGACTTATCTCGAGTGAGATATTGTTGACG,; (SEQ ID NO. 99)CCTCGAGTTATCTCGAGTGAGATATTGTTGACG,; (SEQ ID NO. 100)GCTCGAGTTATCTCGAGTGAGATATTGTTGACG,; (SEQ ID NO. 101)CGTCGAGTTATCTCGAGTGAGATATTGTTGACG,; (SEQ ID NO. 102)GGTGGAGTTATCTCGAGTGAGATATTGTTGACG, and (SEQ ID NO. 103)GCTGGACTTATCTCGAGTGAGATATTGTTGACG,.

The polynucleotides of the present invention may include additionalsequences (e.g. promoters) such that they may be used as expressionconstructs. In addition, the polynucleotides may include sequences whichrender this vector suitable for replication and integration inprokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).Typical cloning vectors contain transcription and translation initiationsequences (e.g., promoters, enhancers) and transcription and translationterminators (e.g., polyadenylation signals), as described herein below.

According to one embodiment, each of the enzyme coding sequences in thepolynucleotide is operatively linked to a promoter. In one embodiment,the identical promoter controls expression of each of the enzymesencoded in the polynucleotide.

According to another embodiment, different promoters control expressionof the enzymes encoded in the polynucleotide.

Examples of particular promoters are described herein below.

According to one embodiment, the expression construct encodes aselectable marker.

As used herein, the term “selectable marker” refers to a gene capable ofexpression in host cell which allows for ease of selection of thosehosts containing an introduced nucleic acid or vector. Examples of suchselectable markers include but are not limited to antimicrobials, (e.g.,kanamycin, erythromycin, actinomycin, chloramphenicol and tetracycline).Thus, the term “selectable marker” refers to genes that provide anindication that a host cell has taken up an exogenous polynucleotidesequence or some other reaction has occurred. Typically, selectablemarkers are genes that confer antimicrobial resistance or a metabolicadvantage on the host cell to allow cells containing the exogenous DNAto be distinguished from cells that have not received any exogenoussequence during the transformation.

As mentioned the polynucleotides described herein encode enzymes whichare part of a biosynthesis pathway of an identical product.

As used herein, the phrase “biosynthesis pathway” or “metabolic pathway”refers to a cellular or a cellular (cell-free) system for converting asubstrate to a product of interest, where the system comprises aplurality of enzymes and may additionally comprise substrates acted uponby one or more of the enzymes, products of the enzyme-catalyzedreactions, co-factors utilized by the enzymes, and the like. The systemmay be present in an intact cell, or in a lysate of a cell.

Many metabolic pathways are known and have been described in microbialsystems, and are accessible in public databases; see, e.g., Smolke, Ed.,The Metabolic Pathway Engineering Handbook: Tools and Applications, CRCPress, New York (2009); Stephanopoulos, Nielsen, and Aristidou, Eds.,Metabolic Engineering: Principles and Methodology, Academic Press, NewYork (1998); Greenberg, Metabolic Pathways: Energetics, TricarboxylicAcid Cycle, and Carbohydrates, Academic Press, New York (1967); and D.M. Greenberg's multi-volume series entitled Metabolic pathways, Volumes1-7, each of which is incorporated herein by reference.

In one embodiment, the pathways direct production of a food product, apharmaceutical or a fuel.

Biosynthesis pathways may include, for example, pathways involved incarbohydrate, amino acid, nucleic acid, steroid, fatty acid, and naturalproduct biosynthesis, and encompass the synthesis of various chemicalcompounds and materials, including, but not limited to:

a) antibiotics; e.g., actinomycin, bleomycin, rifamycin,chloramphenicol, carbapenems, tetracycline, lincomycin, erythromycin,streptomycin, cyclohexamide, puromycin, cycloserine, bacitracin,penicillin, cephalosporin, vancomycin, polymyxin, and gramicidin;

b) bio surfactants; e.g., rhamnolipids, sophorolipids, glycolipids, andlipopeptides;

c) biological fuels; e.g., bioethanol, biodiesel, and biobutanol;

d) amino acids; e.g., L-glutamate, L-lysine, L-phenylalanine, L-asparticacid, L-isoleucine, L-valine, L-tryptophan, L-proline (hydroxyproline),L-threonine, L-methionine, L-tyrosine, and D-p-hydroxyphenylglycine;

e) organic acids; e.g., citric acid, lactic acid, gluconic acid, aceticacid, propionic acid, succinic acid, fumaric acid, and itaconic acid;

f) fatty acids; e.g., arachidonic acid, polyunsaturated fatty acid(PUBA), and .alpha.-linoleic acid;

g) alcohols and polyols; e.g., glycerol, mannitol, erythritol, xylitol,poly-3-hydroxybutyrate, isobutanol, and 1-butanol;

h) flavors and fragrances; e.g., vanillin, benzaldehyde,dihydroxyacetone, 4-(R)-decanolide, and 2-actyl-1-pyrroline;

i) nucleotides; e.g., 5′-guanylic acid and 5′-inosinic acid;

j) vitamins; e.g., vitamin C, vitamin F, vitamin B2, provitamin D2,vitamin B12, folic acid, nicotinamide, biotin, 2-keto-L-gulonic acid,and provitamin Q10;

k) pigments; e.g., astaxanthin, .beta.-carotene, leucopene,monascorubrin, and rubropunctatin;

l) sugars and polysaccharides; e.g., ribose, sorbose, xanthan, gellan,and dextran; and

m) biopolymers and plastics; e.g., polyhydroxyalkanoates (PHA),poly-.gamma.-glutamic acid, and 1,3-propanediol.

Other examples of biosynthesis pathways of interest include thesynthesis of various E. coli metabolites.

A “metabolite” is any substance used or produced during metabolism(e.g., an enzyme, substrate, or product). Herein, a metabolite is often,although not always, the product of an enzyme in the pathway ofinterest.

Exemplary E. coli metabolites include, but are not limited to,2,3-dihydroxybenzoic acid, 2-ketoglutarate, 3-phosphoglycerate,4-hydroxybenzoate, 6-phosphogluconate, acetoacetyl-CoA, acetyl-CoA,acetylphosphate, adenine, adenosine, adenosine phosphosulfate, ADP,ADP-glucose, Alanine, AMP, anthranilate, arginine, Asparagine,Aspartate, ATP, carbamylaspartate, cis-aconitate, citrate, citrulline,CMP, coenzyme A, CTP, cyclic AMP, cytidine, cytosine, dAMP, dATP, dCTP,deoxyadenosine, deoxyguanosine, deoxyribose-5-P, dGMP, dihydroorotate,dihydroxyacetone phosphate, dTDP, dTTP, erythrose-4-phosphate, FAD,flavin mononucleotide, fructose-1,6-bisphosphate, fructose-6-phosphate,fumarate, GDP, gluconate, gluconolactone, glucosamine-6-phosphate,glucose-6-phosphate, glucose-1-phosphate, glutamate, glutamine,glutathione, glutathione disulfide, glyceraldehyde-3-phosphate,glycerate, glycerol-3-phosphate, GMP, GTP, guanine, guanosine,histidine, histidinol, homocysteine, inosine diphosphate, inosinemonophosphate, inosine triphosphate, isoleucine, lysine, malate,malonyl-CoA, methionine, myo-inositol, N-Acetyl-glucosamine-1P,N-acetyl-ornithine, NAD+, NADH, NADP+, NADPH, ornithine, oxaloacetate,phenylalanine, phenylpyruvate, phosphoenolpyruvate, proline,propionyl-CoA, PRPP, pyruvate, quinolinate, riboflavin,ribose-5-phosphate, ribulose-5-phosphate, S-adenosyl-L-methionine,serine, shikimic acid, shikimate, succinate, succinyl-CoA, threonine,tryptophan, tyrosine, UDP, UDP-glucose, UDP-glucuronate,UDP-N-acetylglucosamine, uridine, UTP, valine, and xylulose-5-phosphate.

In certain embodiments, the pathway of interest provides for thesynthesis of shikimic acid and/or shikimate (shikimate is the anionicform of shikimic acid) and synthetic intermediates thereto, anisoprenoid or terpene (e.g., amorphadiene, farnesene, lycopene,astaxanthin, vitamin A, menthol, beta-carotene), poly-3-hydroxybutyrate,isobutanol, and 1-butanol.

A number of reactions may be catalyzed by enzymes in a biosynthesispathway of interest. Broad classes of enzymes, which can be identifiedby enzyme classification number, provided in parentheses, include, butare not limited to:

(EC 1) oxidoreductases; e.g., dehydrogenases, oxidases, reductases,oxidoreductases, synthases, oxygenases, monooxygenases, dioxygenases,lipoxygenases, hydrogenases, transhydrogenases, peroxidases, catalases,epoxidases, hydroxylases, demethylases, desaturases, dismutases,hydroxyltransferases, dehalogenases, and deiodinases;

(EC2) transferases; e.g., transaminases, kinases, dikinases,methyltransferases, hydroxymethyltransferases, formyltransferases,formiminotransferases, carboxytransferases, carbamoyltransferases,amidinotransferases, transaldolases, transketolases, acetyltransferases,acyltransferases palmitoyltransferases, succinyltransferases,malonyltransferases, galloyltransferases, sinapoyltransferases,tigloyltransferases, tetradecanoyltransferases,hydroxycinnamoyltransferases, feruloyltransferases, mycolyltransferases,benzoyltransferases, piperoyltransferases,trimethyltridecanoyltransferase, myristoyltransferases,coumaroyltransferases, thiolases, aminoacyltransferases, phosphorylases,hexosyltransferases, pentosyltransferases, sialyltransferases,pyridinylases, diphosphorylases, cyclotransferases, sulfurylases,adenosyltransferases, carboxyvinyltransferases, isopentenyltransferases,aminocarboxypropyltransferases, dimethylallyltransferases,farnesyltranstransferases, hexaprenyltranstransferases,decaprenylcistransferases, pentaprenyltranstransferases,nonaprenyltransferases, geranylgeranyltransferases,aminocarboxypropyltransferases, oximinotransferases, purinetransferases,phosphodismutases, phosphotransferases, nucleotidyltransferases,polymerases, cholinepho sphotransferases, phosphorylmutases,sulfurtransferases, sulfotransferases, and CoA-transferases;

(EC3) hydrolases; e.g., lipases, esterases, amylases, peptidases,hydrolases, lactonases, deacylases, deacetylases, pheophorbidases,depolymerases, thiolesterases, phosphatases, diphosphatases,triphosphatases, nucleotidases, phytases, phosphodiesterases,phospholipases, sulfatases, cyclases, oligonucleotidases, ribonucleases,exonucleases, endonucleases, glycosidases, nucleosidases, glycosylases,aminopeptidases, dipeptidases, carboxypeptidases,metallocarboxypeptidases, omega-peptidases, serine endopeptidases,cystein endopeptidases, aspartic endopeptidases, metalloendopeptidases,threonine endopeptidases, aminases, amidases, desuccinylases,deformylases, acylases, deiminases, deaminases, dihydrolases,cyclohydrolases, nitrilases, ATPases, GTPases, halidases, dehalogenases,and sulfohydrolases;

(EC 4) lyases; e.g., decarboxylases, carboxylases, carboxykinases,aldolases, epoxylyases, oxoacid-lyases, carbon-carbon lyases,dehydratases, hydratases, synthases, endolyases, exolyases,ammonia-lyases, amidine-lyases, amine-lyases, carbon-sulfur lyases,carbon-halide lyases, phosphorus-oxygen lyases, and dehydrochlorinases;

(EC 5) isomerases; e.g., isomerases, racemases, mutases, tautomerases,phosphomutases, phosphoglucomutases, aminomutases, cycloisomerase,cyclases, topoisomerases; and

(EC 6) ligases; e.g., synthetases, tNRA-ligases, acid-thiol ligases,amide synthases, peptide synthases, cycloligases, carboxylases,DNA-ligases, RNA-ligases, and cyclases.

More specific classes of enzymes include, without limitation,sub-classes of oxidoreductases, transferases, lyases, isomerases, andligases, as provided below. Exemplary oxidoreductases include, but arenot limited to:

(EC 1.1) oxidoreductases acting on the CH—OH group of donors, and anacceptor;

(EC 1.2) oxidoreductases acting on the aldehyde or oxo group of donors,and an acceptor;

(EC 1.3) oxidoreductases acting on the CH—CH group of donors, and anacceptor;

(EC 1.4) oxidoreductases acting on the CH—NH2 group of donors, and anacceptor;

(EC 1.5) oxidoreductases acting on the CH—NH group of donors, and anacceptor;

(EC 1.6) oxidoreductases acting on NADH or NADPH, and an acceptor;

(EC 1.7) oxidoreductases acting on other nitrogenous compounds asdonors, and an acceptor;

(EC 1.8) oxidoreductases acting on a sulfur group of donors, and anacceptor;

(EC 1.9) oxidoreductases acting on a heme group of donors, and anacceptor;

(EC 1.1) oxidoreductases acting on diphenols and related substances asdonors, and an acceptor;

(EC 1.11) oxidoreductases acting on a peroxide as acceptor;

(EC 1.12) oxidoreductases acting on hydrogen as donor, and an acceptor;

(EC 1.13) oxidoreductases acting on single donors with incorporation ofmolecular oxygen, incorporating one or two oxygen atoms;

(EC 1.14) oxidoreductases acting on paired donors, with incorporation orreduction of molecular oxygen, with the donor being 2-oxoglutarate,NADH, NADPH, reduced flavin, flavoprotein, pteridine, iron-sulfurprotein, ascorbate;

(EC 1.15) oxidoreductases acting on superoxide radicals as acceptor;

(EC 1.16) oxidoreductases oxidizing metal ions, and an acceptor;

(EC 1.17) oxidoreductases acting on CH or CH2 groups, and an acceptor;

(EC 1.18) oxidoreductases acting on iron-sulfur proteins as donors, andan acceptor;

(EC 1.19) oxidoreductases acting on reduced flavodoxin as donor, and anacceptor;

(EC 1.2) oxidoreductases acting on phosphorus or arsenic in donors, andan acceptor; and

(EC 1.21) oxidoreductases acting on X—H and Y—H to form an X—Y bond, andan acceptor; where acceptors for each donor category may include,without limitation: NAD, NADP, heme protein, oxygen, disulfide, quinone,an iron-sulfur protein, a flavin, a nitrogenous group, a cytochrome,dinitrogen, and H+.

Exemplary transferases include, but are not limited to:

(EC 2.1) transferases transferring one-carbon groups;

(EC 2.2) transferases transferring aldehyde or ketonic groups;

(EC 2.3) Acyltransferases;

(EC 2.4) Glycosyltransferases;

(EC 2.5) transferases transferring alkyl or aryl groups, other thanmethyl groups;

(EC 2.6) transferases transferring nitrogenous groups;

(EC 2.7) transferases transferring phosphorus-containing groups;

(EC 2.8) transferases transferring sulfur-containing groups; and

(EC 2.9) transferases transferring selenium-containing groups.

Exemplary hydrolases include, but are not limited to:

(EC 3.1) hydrolases acting on ester bonds;

(EC 3.2) Glycosylases;

(EC 3.3) hydrolases acting on ether bonds;

(EC 3.4) hydrolases acting on peptide bonds (peptidases);

(EC 3.5) hydrolases acting on carbon-nitrogen bonds, other than peptidebonds;

(EC 3.6) hydrolases acting on acid anhydrides;

(EC 3.7) hydrolases acting on carbon-carbon bonds;

(EC 3.8) hydrolases acting on halide bonds;

(EC 3.9) hydrolases acting on phosphorus-nitrogen bonds;

(EC 3.1) hydrolases acting on sulfur-nitrogen bonds;

(EC 3.11) hydrolases acting on carbon-phosphorus bonds;

(EC 3.12) hydrolases acting on sulfur-sulfur bonds; and (EC 3.13)hydrolases acting on carbon-sulfur bonds.

Exemplary lyases include, but are not limited to:

(EC 4.1) Carbon-carbon lyases;

(EC 4.2) Carbon-oxygen lyases;

(EC 4.3) Carbon-nitrogen lyases;

(EC 4.4) Carbon-sulfur lyases;

(EC 4.5) Carbon-halide lyases; and

(EC 4.6) Phosphorus-oxygen lyases.

Exemplary isomerases include, but are not limited to:

(EC 5.1) Racemases and epimerases;

(EC 5.2) cis-trans-Isomerases;

(EC 5.3) Intramolecular isomerases;

(EC 5.4) Intramolecular transferases (mutases); and

(EC 5.5) Intramolecular lyases.

Exemplary ligases include, but are not limited to:

(EC 6.1) ligases forming carbon-oxygen bonds;

(EC 6.2) ligases forming carbon-sulfur bonds;

(EC 6.3) ligases forming carbon-nitrogen bonds;

(EC 6.4) ligases forming carbon-carbon bonds;

(EC 6.5) ligases forming phosphoric ester bonds; and

(EC 6.6) ligases forming nitrogen-metal bonds.

Isozymes (also known as isoenzymes) are enzymes that differ in aminoacid sequence but catalyze the same chemical reaction. At some points ina pathway of interest, two or more isozymes may be present. Isozymes maydisplay different kinetic parameters, and/or different regulatoryproperties.

Enzymes involved in a pathway of interest or associated pathway may alsobe classified according to the role of the enzyme. Direct involvementenzymes (class 1) in a cell or cell lysate catalyze a reaction in thepathway. It is typical of pathways that such direct enzymes are one of achain, where a product of a first enzyme is the substrate of a secondenzyme, the product of the second enzyme is the substrate of a thirdenzyme, and so forth, which eventually results in the product ofinterest. Indirect involvement enzymes (class 2) in a cell or celllysate react in an associated pathway, usually in the production of asubstrate used in the pathway of interest.

Pathways of interest for use in the methods of the described herein willusually comprise at least one enzyme, at least two enzymes, at leastthree enzymes, at least four enzymes, or more, e.g., between 1 to 50enzymes, between 1 to 40 enzymes, between 1 to 30 enzymes, between 1 to20 enzymes, between 1 to 10 enzymes, between 1 to 5 enzymes, between 1to 2 enzymes, between 2 to 50 enzymes, between 2 to 40 enzymes, between2 to 30 enzymes, between 2 to 20 enzymes, between 2 to 10 enzymes,between 2 to 5 enzymes, between 2 to 4 enzymes, between 5 to 50 enzymes,between 5 to 40 enzymes, between 5 to 30 enzymes, between 5 to 20enzymes, between 5 to 10 enzymes, between 5 to 8 enzymes, between 10 to50 enzymes, between 10 to 40 enzymes, between 10 to 30 enzymes, orbetween 10 to 20 enzymes, inclusive.

Enzymes in a pathway may be naturally occurring, or modified to optimizea particular characteristic of interest, e.g., substrate specificity,reaction kinetics, solubility, and/or insensitivity to feedbackinhibition. In addition, in some cases, the gene expressing the enzymewill be optimized for codon usage within the host cell. In someembodiments, the complete pathway comprises enzymes from a singleorganism, however such is not required, and combining enzymes frommultiple organisms is also contemplated. For some purposes, a pathwaymay be endogenous to the host cell, but such is also not required, and acomplete pathway or components of a pathway may be introduced into ahost cell. Where the system is provided in an intact cell, the completeset of enzymes of the pathway of interest can be present in the cell.

It should be appreciated that the genes encoding the enzymes associatedwith the embodiments described herein can be obtained from a variety ofsources. As one of ordinary skill in the art would be aware, homologousgenes for these targeted enzymes exist in many species and can beidentified by homology searches, for example through a protein BLASTsearch, available at the NCBI internet site(ncbi(dot)nlm(doy)nih(dot)gov). Genes encoding these enzymes can bePCR-amplified from DNA from any source which contains the given enzyme,for example using degenerate primers, as would be understood by one ofordinary skill in the art. In some embodiments, the gene encoding agiven enzyme can be synthetic (artificial), for example, DNA synthesizedfrom sugars, nitrogen-based compounds, phosphates, and othercompound/reagents required for DNA synthesis. Any means of obtaining thegenes encoding for the enzymes discussed here are compatible withaspects of the embodiments described herein.

Products of the pathway may be stable or relatively labile, but in someinstances the final product is sufficiently stable that it can beisolated from the cell, cell lysate, or reaction mixture.

The amount of product produced in a reaction can be measured in variousways, for example, by enzymatic assays which produce a colored orfluorometric product or by high-performance liquid chromatography (HPLC)methods. In certain embodiments, the product is measured utilizing anassay which measures the activity or concentration of the particularproduct being produced. If the product is a protein, it may bequantified on the RNA or protein level.

One skilled in the art is well aware of methods for introducingpolynucleotides into host cells and particularly into E. coli, Bacillusand Pantoea host cells. General transformation techniques are disclosedin CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Vol. 1, eds. Ausubel et al.John Wiley & Sons Inc, (1987) Chap. 7. and Sambrook, J., et al.,MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor LaboratoryPress (1989). Reference is also made to Ferrari et al., Genetics pgs57-72 in Hardwood et al. Ed. BACILLUS, Plenum Publishing Corp. 1989;Chang et al., (1979) Mol. Gen. Genet. 168:11-15; Smith et al., (1986)Appl. and Env. Microbiol. 51:634 and Potter, H. (1988) Anal Biochem174:361-373 wherein methods of transformation, includingelectroporation, protoplast transformation and congression; transductionand protoplast fusion are disclosed. Methods of transformations areparticularly preferred. Methods suitable for the maintenance and growthof bacterial cells is well known and reference is made to the Manual ofMethods of General Bacteriology, Eds. P. Gerhardt et al., AmericanSociety for Microbiology, Washington, D.C. (1981) and T. D. Brock inBiotechnology: A Textbook of Industrial Microbiology 2 ed. (1989)Sinauer Associates, Sunderland Mass.

As used herein, the term “introduced” used in the context of inserting anucleic acid sequence into a cell, means “transfection,”“transformation,” or “transduction,” and includes reference to theincorporation of a nucleic acid sequence into a eukaryotic orprokaryotic cell where the nucleic acid sequence may be incorporatedinto the genome of the cell (e.g., chromosome, plasmid, plastid, ormitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (for example, transfected mRNA).

As used herein, the terms “transformed,” “stably transformed,” and“transgenic” used in reference to a cell means the cell has a non-native(heterologous) nucleic acid sequence integrated into its genome or as anepisomal plasmid that is maintained through two or more generations.

The transformed host cells are selected based on the phenotype responseto a selectable marker which was provided in an insertion DNA construct.In some embodiments the selectable marker may be excised out of the hostcell. (Cherepanov et al. (1995) Gene 158:9-14).

Host cells for pathway engineering include a wide variety ofheterotrophic and autotrophic microorganisms, including, but not limitedto, bacteria, fungi and protozoans. In certain embodiments, host cellsinclude those for which means by which a polypeptide can be directed toa cellular compartment or extracellular compartments are known. In someembodiments, the cell is any type of cell that recombinantly expressesany one or more of the nucleic acids described herein. Such cellsinclude prokaryotic and eukaryotic cells. In some embodiments, the cellis a bacterial cell, such as Escherichia spp., Streptomyces spp.,Zymonas spp., Acetobacter spp., Citrobacter spp., Synechocystis spp.,Rhizobium spp., Clostridium spp., Corynebacterium spp., Streptococcusspp., Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillusspp., Alcaligenes spp., Pseudomonas spp., Aeromonas spp., Azotobacterspp., Comamonas spp., Mycobacterium spp., Rhodococcus spp.,Gluconobacter spp., Ralstonia spp., Acidithiobacillus spp., Microlunatusspp., Geobacter spp., Geobacillus spp., Arthrobacter spp.,Flavobacterium spp., Serratia spp., Saccharopolyspora spp., Thermusspp., Stenotrophomonas spp., Chromobacterium spp., Sinorhizobium spp.,Saccharopolyspora spp., Agrobacterium spp. and Pantoea spp. Thebacterial cell can be a Gram-negative cell such as an Escherichia coli(E. coli) cell, or a Gram-positive cell such as a species of Bacillus.In other embodiments the cell is a fungal cell such as yeast cells,e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffiaspp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomycesspp., Pachysolen spp., Debaryomyces spp., Yarrowia spp. and industrialpolyploid yeast strains. Other non-limiting examples of fungi includeAspergillus spp., Pennicilium spp., Fusarium spp., Rhizopus spp.,Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp.,Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp. Inother embodiments, the cell is an algal cell, a plant cell, or amammalian cell. It should be appreciated that some cells compatible withthe embodiments described herein may express an endogenous copy of oneor more of the genes described herein as well as a recombinant copy.Species of interest include, without limitation, S. cerevisiae, E. coli,Pseudomonas species, Klebsiella species, and Synechocystis species.

Following selection of the appropriate RBS enzyme combination in aparticular pathway, the polynucleotides may be introduced into a cellsystem as separate expression vector or on a single expression vector soas to prepare commercial quantities of that product. The final productmay then be isolated. A variety of methodologies well known to theskilled practitioner can be utilized to obtain isolated polypeptidesassociated with the embodiments described herein. The polypeptide may bepurified from cells by immunochromatography, HPLC, size exclusionchromatography, ion exchange chromatography and immune affinitychromatography.

The reactions may utilize a large scale reactor, small scale, or may bemultiplexed to perform a plurality of simultaneous syntheses. Continuousreactions will use a feed mechanism to introduce a flow of reagents, andmay isolate the end-product as part of the process. Batch systems arealso of interest, where additional reagents may be introduced over timeto prolong the period of time for active synthesis. A reactor may be runin any mode such as batch, extended batch, semi-batch, semi-continuous,fed-batch, and continuous, and which will be selected in accordance withthe application purpose.

The reactions may be of any volume, either in a small scale (e.g.,usually at least about 1 ml and not more than about 15 ml) or in ascaled up reaction (e.g., where the reaction volume is at least about 15ml, usually at least about 50 ml, more usually at least about 100 ml,and may be 500 ml, 1000 ml, or greater up to many thousands of liters ofvolume). Reactions may be conducted at any scale.

Using the strategy described herein, the present inventors haveuncovered the optimal RBS-enzyme combination for the synthesis ofastaxanthin.

Thus, according to another aspect of the present invention there isprovided a method of generating astaxanthin comprising expressingpolynucleotides encoding enzymes of an astaxanthin pathway, thepolynucleotides comprising:

(i) a polynucleotide which encodes Phytoene dehydrogenase (crtI) and afirst transcriptional regulatory sequence;

(ii) a polynucleotide which encodes Beta-lycopene cyclase (lcy-B) and asecond transcriptional regulatory sequence;

(iii) a polynucleotide which encodes Beta-carotene ketolase (crtW) and athird transcriptional regulatory sequence;

wherein the first, second and third regulatory sequence are selectedsuch that the expression of the lcy-B and the crtW is greater than alevel of expression of the crtI.

The method of the present invention contemplates expression of at leastone, at least two, at least three at least four additionalpolynucleotides encoding enzymes of the astaxanthin pathway.

Additional polynucleotides include:

(iv) a polynucleotide which encodes Isopentenyl pyrophosphate (idi) anda fourth transcriptional regulatory sequence;

(v) a polynucleotide which encodes Geranylgeranyl pyrophosphate synthase(crtE) and a fifth transcriptional regulatory sequence;

(vi) a polynucleotide which encodes Prephytoene pyrophosphate synthase(crtB) and a sixth transcriptional regulatory sequence;

and

(vii) a polynucleotide which encodes Beta-carotene hydroxylase (crtZ)and a seventh transcriptional regulatory sequence.

As used herein, the term “astaxanthin” refers to any one of its threestereoisomers (3R,3′R), (3R,3'S) (meso) and (3S,3'S), also known as3,3′-dihydroxy-β-carotene-4,4′-dione, having a molecular formulaC₄₀H₅₂O₄.

In order to generate astaxanthin in biological cells, the cellspreferably express enzymes of the astaxanthin pathway.

Exemplary enzymes are listed herein below.

Geranylgeranyl Pyrophosphate Synthase (crtE or GGPP Synthase):

The term “crtE” refers to an enzyme which is capable of convertingfarnesyl diphosphate (FPP) to geranylgeranyl diphosphate GGPP (EC2.5.1.29). GenBank Accession Nos. of non-limiting examples of crtE arelisted below and in Table 15 of U.S. Patent Application No. 20110039299,incorporated herein by reference. A crtE of the present invention alsorefers to homologs (e.g., polypeptides which are at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 87%, at least 89%, at least 91%, at least93%, at least 95% or more say 100% homologous to crtE sequences listedherein below as determined using BlastP software of the National Centerof Biotechnology Information (NCBI) using default parameters). Thehomolog may also refer to a deletion, insertion, or substitutionvariant, including an amino acid substitution, thereof and biologicallyactive polypeptide fragments thereof.

Illustrative examples of nucleotide sequences for geranylgeranylpyrophosphate synthase include but are not limited to: (ATHGERPYRS;Arabidopsis thaliana), (BT005328; Arabidopsis thaliana), (NM_119845;Arabidopsis thaliana), (NZ_AAJM01000380, Locus ZP_00743052; Bacillusthuringiensis serovar israelensis, ATCC 35646 sq1563), (CRGGPPS;Catharanthus roseus), (NZ_AABF02000074, Locus ZP.sub.—00144509;Fusobacterium nucleatum subsp. vincentii, ATCC 49256), (GFGGPPSGN;Gibberella fujikuroi), (AY371321; Ginkgo biloba), (AB055496; Heveabrasiliensis), (AB017971; Homo sapiens), (MCI276129; Mucorcircinelloides f. lusitanicus), (AB016044; Mus musculus), (AABX01000298,Locus NCU01427; Neurospora crassa), (NCU20940; Neurospora crassa),(NZ_AAKL01000008, Locus ZP.sub.—00943566; Ralstonia solanacearum UW551),(AB118238; Rattus norvegicus), (SCU31632; Saccharomyces cerevisiae),(AB016095; Synechococcus elongates), (SAGGPS; Sinapis alba), (SSOGDS;Sulfolobus acidocaldarius), (NC_007759, Locus YP_461832; Syntrophusaciditrophicus SB), and (NC.sub.—006840, Locus YP.sub.—204095; Vibriofischeri ES114). According to a specific embodiment, the GGPP synthasecomprises a sequence derived from Pantoea agglomerans—GenBank:AAA21260.1 (SEQ ID NO: 62).

Prephytoene Pyrophosphate Synthase (crtB):

As used herein, the term “crtB” refers to the enzyme which converts GGPPto phytoene (EC=2.5.1.32), also known as phytoene synthase. GenBankAccession Nos. of non-limiting examples of crtB are listed in Table 18of U.S. Patent Application No. 20110039299, incorporated herein byreference. A crtB of the present invention also refers to homologs(e.g., polypeptides which are at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 87%, at least 89%, at least 91%, at least 93%, at least 95% ormore say 100% homologous to crtE sequences listed in Table 18 of U.S.Patent Application No. 20110039299 as determined using BlastP softwareof the National Center of Biotechnology Information (NCBI) using defaultparameters). The homolog may also refer to a deletion, insertion, orsubstitution variant, including an amino acid substitution, thereof andbiologically active polypeptide fragments thereof.

According to a specific embodiment, the crtB comprises a sequencederived from Pantoea agglomerans—GenBank: AAA21264.1 (SEQ ID NO: 63).

Phytoene Dehydrogenase (crtI):

As used herein, the term “crtI” refers to the enzyme which convertsphytoene to lycopene (EC=1.14.99). GenBank Accession Nos. ofnon-limiting examples of crtI are listed in Tables 17A and 17B of U.S.Patent Application No. 20110039299, incorporated herein by reference. AcrtE of the present invention also refers to homologs (e.g.,polypeptides which are at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 87%, at least 89%, at least 91%, at least 93%, at least 95% ormore say 100% homologous to crtI sequences listed in Tables 17A and 17Bof U.S. Patent Application No. 20110039299 as determined using BlastPsoftware of the National Center of Biotechnology Information (NCBI)using default parameters). The homolog may also refer to a deletion,insertion, or substitution variant, including an amino acidsubstitution, thereof and biologically active polypeptide fragmentsthereof.

According to a specific embodiment, the crtI comprises a sequencederived from Pantoea agglomerans—GenBank: AAA21263.1 (SEQ ID NO: 64).

Isopentenyl Pyrophosphate Isomerase (Idi):

As used herein, the term “idi” refers to the isomerase enzyme whichcatalyzes the conversion of the relatively un-reactive isopentenylpyrophosphate (IPP) to the more-reactive electrophile dimethylallylpyrophosphate (DMAPP). (EC=5.3.3.2). It is also known asIsopentenyl-diphosphate delta isomerase. GenBank Accession Nos. ofnon-limiting examples of idi are listed in Table 13 of U.S. PatentApplication No. 20110039299, incorporated herein by reference. A idi ofthe present invention also refers to homologs (e.g., polypeptides whichare at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 87%, at least89%, at least 91%, at least 93%, at least 95% or more say 100%homologous to idi sequences listed in Table 13 of U.S. PatentApplication No. 20110039299 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters). The homolog may also refer to a deletion, insertion, orsubstitution variant, including an amino acid substitution, thereof andbiologically active polypeptide fragments thereof.

According to a specific embodiment, the idi comprises a sequence derivedfrom Haematococcus pluvialis—GenBank: AAC32208.1 (SEQ ID NO: 65).

Beta-Lycopene Cyclase (lcy-B):

As used herein, the term “lcy-B” refers to the enzyme which catalyzesthe conversion of lycopene to carotene. GenBank Accession Nos. ofnon-limiting examples of lcy-B are listed in Table 23 of U.S. PatentApplication No. 20110039299, incorporated herein by reference. A lcy-Bof the present invention also refers to homologs (e.g., polypeptideswhich are at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 87%, atleast 89%, at least 91%, at least 93%, at least 95% or more say 100%homologous to lcy-B sequences listed in Table 23 of U.S. PatentApplication No. 20110039299 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters). The homolog may also refer to a deletion, insertion, orsubstitution variant, including an amino acid substitution, thereof andbiologically active polypeptide fragments thereof.

According to a specific embodiment, the lcy-B comprises a sequencederived from Solanum lycopersicum—GenBank: ABR57232.1 (SEQ ID NO: 66).

Beta-Carotene Hydroxylase (crtZ):

As used herein, the term “crtZ” refers to the enzyme which catalyzes theconversion of carotene to Zeaxanthin and/or the enzyme which catalyzesthe conversion of canthaxanthin to astaxanthin (EC 1.14.13). GenBankAccession Nos. of non-limiting examples of crtZ are listed in Table 20of U.S. Patent Application No. 20110039299, incorporated herein byreference. A crtZ of the present invention also refers to homologs(e.g., polypeptides which are at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 87%, at least 89%, at least 91%, at least 93%, at least 95% ormore say 100% homologous to crtZ sequences listed in Table 20 of U.S.Patent Application No. 20110039299 as determined using BlastP softwareof the National Center of Biotechnology Information (NCBI) using defaultparameters). The homolog may also refer to a deletion, insertion, orsubstitution variant, including an amino acid substitution, thereof andbiologically active polypeptide fragments thereof.

According to a specific embodiment, the crtZ comprises a sequencederived from Pantoea ananatis—Swiss-Prot: P21688.1 (SEQ ID NO: 67).

Beta-Carotene Ketolase (crtW):

As used herein, the term “crtW” refers to the enzyme which catalyzes theconversion of carotene to canthaxanthin and/or the enzyme whichcatalyzes the conversion of zeaxanthin to astaxanthin. GenBank AccessionNos. of non-limiting examples of crtW are listed in Table 19 of U.S.Patent Application No. 20110039299, incorporated herein by reference. AcrtW of the present invention also refers to homologs (e.g.,polypeptides which are at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 87%, at least 89%, at least 91%, at least 93%, at least 95% ormore say 100% homologous to crtW sequences listed in Table 19 of U.S.Patent Application No. 20110039299 as determined using BlastP softwareof the National Center of Biotechnology Information (NCBI) using defaultparameters). The homolog may also refer to a deletion, insertion, orsubstitution variant, including an amino acid substitution, thereof andbiologically active polypeptide fragments thereof. According to aspecific embodiment, the crtW comprises a sequence derived from Nostocsphaeroides—GenBank: BAB74888.1 (SEQ ID NO: 68).

Preferably, the cells also express 1-deoxyxylulose-5-phosphate synthase(dxs).

As used herein the term “1-deoxyxylulose-5-phosphate synthase (dxs)”refers to the enzyme which catalyzes the condensation of pyruvate andD-glyceraldehyde 3-phosphate to D-1-deoxyxylulose 5-phosphate (DOXP).(EC 2.2.1.7). Exemplary accession numbers include those provided inTable 3 of U.S. Patent Application No. 20040268436, incorporated hereinby reference. A dxs of the present invention also refers to homologs(e.g., polypeptides which are at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 87%, at least 89%, at least 91%, at least 93%, at least 95% ormore say 100% homologous to dxs sequences listed in Table 3 of U.S.Patent Application No. 20040268436 as determined using BlastP softwareof the National Center of Biotechnology Information (NCBI) using defaultparameters). The homolog may also refer to a deletion, insertion, orsubstitution variant, including an amino acid substitution, thereof andbiologically active polypeptide fragments thereof. According to aspecific embodiment, the dxs comprises a sequence derived fromEscherichia coli—Swiss-Prot: A7ZX72.1 (SEQ ID NO: 69).

Additional enzymes of the astaxanthin pathway which may also beexpressed in the host system are described in U.S. Patent ApplicationNo. 20110039299, incorporated herein by reference.

A variety of prokaryotic or eukaryotic cells or eukaryotic cells can beused as host-expression systems to express the polypeptides of thepresent invention. These include, but are not limited to,microorganisms, such as bacteria transformed with a recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorcontaining the polypeptide coding sequence; mammalian expression systemssuch as CHO cells, fungi such as yeast transformed with recombinantyeast expression vectors containing the polypeptide coding sequence;algae and plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors, such asTi plasmid, containing the polypeptide coding sequences. Additional hostexpression systems are further described herein above.

According to a preferred embodiment, the enzymes of the astaxanthinpathway are expressed in bacterial cells.

In one embodiment, the host cell is a bacterial cell such as a grampositive bacteria. In another embodiment the host cell is agram-negative bacteria. In some preferred embodiments, the term refersto cells in the genus Pantoea, the genus Bacillus and E. coli cells.

As used herein, “the genus Bacillus” includes all members known to thoseof skill in the art, including but not limited to B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B.megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.It is recognized that the genus Bacillus continues to undergotaxonomical reorganization. Thus, it is intended that the genus includespecies that have been reclassified, including but not limited to suchorganisms as B. stearothermophilus, which is now named “Geobacillusstearothermophilus.” The production of resistant endospores in thepresence of oxygen is considered the defining feature of the genusBacillus, although this characteristic also applies to the recentlynamed Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus,Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus,Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, andVirgibacillus.

As used herein, “the genus Pantoea” includes all members known to thoseof skill in the art, including but not limited to P. agglomerans, P.dispersa, P. punctata, P. citrea, P. terrea, P. ananas and P. sterartii.It is recognized that the genus Pantoea continues to undergo taxonomicalreorganization. Thus, it is intended that the genus include species thathave been reclassified, including but not limited to such organisms asErwinia herbicola.

The polynucleotide sequences of the present invention are inserted intoexpression vectors (i.e., a nucleic acid construct) to enable expressionof the polypeptides of the astaxanthin pathway in the cells.

The number of enzymes that need to be expressed using expression vectorsin a particular cell system will depend on the number of those enzymeswhich are endogenously expressed in that particular cell system. Thus,for example if a particular cell system endogenously expresses crtW to asufficient extent, that enzyme may not need to be exogenously expressedusing recombinant methods.

The present invention contemplates inserting a polynucleotide sequenceencoding one, two, three, four, five, six, seven or eight of thepolypeptides of the astaxanthin pathway on a single expression vector.Thus, the expressing of the polypeptides may be effected by introducinga single expression vector encoding all the necessary enzymes or aplurality of vectors encoding various combinations of the polypeptidesof the astaxanthin pathway.

The present inventors have found that for optimum expression ofastaxanthin, the transcriptional regulatory elements operatively linkedto the polynucleotides encoding the components of the astaxanthinpathway should be selected to ensure a particular ratio of expression.

According to a particular embodiment, the expression of both Icy-B andcrtW should be manipulated such that their expression level is higherthan the level of expression of crtI. Preferably the level of expressionof both Icy-B and crtW is at least two times the level of expression ofcrtI. According to another embodiment, the level of expression of bothIcy-B and crtW is at least five times the level of expression of crtI.

According to another embodiment, the level of expression of both Icy-Band crtW is at least ten times the level of expression of crtI.According to another embodiment, the level of expression of both Icy-Band crtW is at least 20 times the level of expression of crtI. Accordingto another embodiment, the level of expression of both Icy-B and crtW isat least 50 times the level of expression of crtI. According to anotherembodiment, the level of expression of both Icy-B and crtW is at least100 times the level of expression of crtI.

As used herein, the phrase “transcriptional regulatory element” refersto a sequence of bases operatively linked to the protein coding regionof the polynucleotide which controls transcription thereof.

Examples of transcriptional regulatory elements include, but are notlimited to a promoter, an enhancer, an mRNA stability effecting sequenceand a ribosomal binding site (RBS).

For the purposes of this application, a “promoter” or “promoter region”is a nucleic acid sequence that is recognized and bound by a DNAdependent RNA polymerase during initiation of transcription. Thepromoter, together with other transcriptional and translationalregulatory elements is necessary to express a given gene or group ofgenes (an operon). The promoter may be a regulatable promoter, such asPtrc, which is induced by IPTG or a constitutive promoter.

Promoter sequences useful for creating artificial promoters according tothe invention include the precursor promoters listed in Table 1 below.All promoters in the table are characterized with respect to thebeta-lactamase promoter Pbla and promoter strengths are given in“Pbla-units”. (Deuschle et al., EMBO Journal 5(11):2987-2994 (1986)).Further examples of promoters and nucleic acid sequences thereof areprovided in U.S. Patent Application No. 20120015849, incorporated hereinby reference. In general, promoters useful in the invention includepromoter sequences of between 200 to 20 base pairs (bp), preferably 150to 25 bp, more preferably between 100 to 30 bp and most preferablybetween 50 to 30 bp upstream from the transcription start site (+1).

Additional promoters useful in the invention are disclosed in Sommer etal., (2000) Microbiol. 146:2643-2653, wherein the sequence of Ptac andvariants containing 1 or 2 base pair changes are taught.

TABLE 1 Relative Promoter Source activity B-lactamase (bla) E. coli 1P-Consensus (con) Synthetic DNA 4 PTac1 (Trc) Hybrid of 2 promoters 17PLacUV5 Mutant of Lac 3.3 Plac E. coli LacZ gene 5.7 PL Phage λ 37 PA1Phage T7 22 PA2 Phage T7 20 PA3 Phage T7 76 PJ5 Phage T3 9 PG25 Phage T319 PN25 Phage T3 30 PD/E20 Phage T3 56 PH207 Phage T3 55

Promoter strength can be quantified using in vitro methods that measurethe kinetics of binding of the RNA polymerase to a particular piece ofDNA, and also allows the measurement of transcription initiation (HawleyD. K et al., Chapter 3: in: PROMOTERS: STRUCTURE AND FUNCTION. R.L/Rodriguez and M. J. Chamberlin eds. Praeger Scientific. New York).Further, promoter strength can be quantified by linking the promoter toa polynucleotide sequence encoding a detectable protein (e.g. afluorescent marker), as further described herein below. In vivo methodshave been used also to quantify promoter strength. In this case, theapproach has been to fuse the promoter to a reporter gene and theefficiency of RNA synthesis measured.

As mentioned, the transcriptional regulatory element may comprise aribosome binding site (RBS), which are further described herein above.

According to a particular embodiment, the RBS linked to crtI is onewhich brings about expression of a nucleic acid sequence to at least60%, at least 70%, at least 80%, at least 90% of the extent as the RBShaving a sequence as set forth in SEQ ID NO: 4, 5, 6 or 7 for the samenucleic acid sequence under identical experimental and transcriptionalconditions (i.e. in the same cell, for the same time, at the sametemperature, with the same promoter etc.).

According to a particular embodiment, the RBS linked to lcyB is onewhich brings about expression of a nucleic acid sequence to at least60%, at least 70%, at least 80%, at least 90% of the extent as the RBShaving a sequence as set forth in SEQ ID NO: 2 or 3 for the same nucleicacid sequence under identical experimental and transcriptionalconditions (i.e. in the same cell, with the same promoter etc.).

According to a particular embodiment, the RBS linked to crtW is onewhich brings about expression of a nucleic acid sequence to at least60%, at least 70%, at least 80%, at least 90% of the extent as the RBShaving a sequence as set forth in SEQ ID NO: 2 or 3 for the same nucleicacid sequence under identical experimental and transcriptionalconditions (i.e. in the same cell, with the same promoter etc.).

One particular combination of sequences contemplated by the presentinventors is as follows:

The RBS linked to the polynucleotide sequence encoding idi is one whichbrings about expression of a nucleic acid sequence to at least 60%, atleast 70%, at least 80%, at least 90% of the extent as the RBS having asequence as set forth in SEQ ID NO: 5 for the same nucleic acid sequenceunder identical experimental and transcriptional conditions (i.e. in thesame cell, with the same promoter etc.).

The RBS linked to the polynucleotide sequence encoding crtE is one whichbrings about expression of a nucleic acid sequence to at least 60%, atleast 70%, at least 80%, at least 90% of the extent as the RBS having asequence as set forth in SEQ ID NO: 5 for the same nucleic acid sequenceunder identical experimental and transcriptional conditions (i.e. in thesame cell, with the same promoter etc.).

The RBS linked to the polynucleotide sequence encoding crtB is one whichbrings about expression of a nucleic acid sequence to at least 60%, atleast 70%, at least 80%, at least 90% of the extent as the RBS having asequence as set forth in SEQ ID NO: 3 for the same nucleic acid sequenceunder identical experimental and transcriptional conditions (i.e. in thesame cell, with the same promoter etc.).

The RBS linked to the polynucleotide sequence encoding crtI is one whichbrings about expression of a nucleic acid sequence to at least 60%, atleast 70%, at least 80%, at least 90% of the extent as the RBS having asequence as set forth in SEQ ID NO: 7 for the same nucleic acid sequenceunder identical experimental and transcriptional conditions (i.e. in thesame cell, with the same promoter etc.).

The RBS linked to the polynucleotide sequence encoding lcy-B is onewhich brings about expression of a nucleic acid sequence to at least60%, at least 70%, at least 80%, at least 90% of the extent as the RBShaving a sequence as set forth in SEQ ID NO: 2 for the same nucleic acidsequence under identical experimental and transcriptional conditions(i.e. in the same cell, with the same promoter etc.).

The RBS linked to the polynucleotide sequence encoding crtW is one whichbrings about expression of a nucleic acid sequence to at least 60%, atleast 70%, at least 80%, at least 90% of the extent as the RBS having asequence as set forth in SEQ ID NO: 3 for the same nucleic acid sequenceunder identical experimental and transcriptional conditions (i.e. in thesame cell, with the same promoter etc.).

The RBS linked to the polynucleotide sequence encoding crtZ is one whichbrings about expression of a nucleic acid sequence to at least 60%, atleast 70%, at least 80%, at least 90% of the extent as the RBS having asequence as set forth in SEQ ID NO: 6 for the same nucleic acid sequenceunder identical experimental and transcriptional conditions (i.e. in thesame cell, with the same promoter etc.).

Another exemplary combination of sequences is as follows:

The RBS linked to the polynucleotide sequence encoding idi is one whichbrings about expression of a nucleic acid sequence to at least 60%, atleast 70%, at least 80%, at least 90% of the extent as the RBS having asequence as set forth in SEQ ID NO: 6 for the same nucleic acid sequenceunder identical transcriptional conditions (i.e. in the same cell, withthe same promoter etc.).

The RBS linked to the polynucleotide sequence encoding crtE is one whichbrings about expression of a nucleic acid sequence to at least 60%, atleast 70%, at least 80%, at least 90% of the extent as the RBS having asequence as set forth in SEQ ID NO: 3 for the same nucleic acid sequenceunder identical experimental and transcriptional conditions (i.e. in thesame cell, with the same promoter etc.).

The RBS linked to the polynucleotide sequence encoding crtB is one whichbrings about expression of a nucleic acid sequence to at least 60%, atleast 70%, at least 80%, at least 90% of the extent as the RBS having asequence as set forth in SEQ ID NO: 7 for the same nucleic acid sequenceunder identical experimental and transcriptional conditions (i.e. in thesame cell, with the same promoter etc.).

The RBS linked to the polynucleotide sequence encoding crtI is one whichbrings about expression of a nucleic acid sequence to at least 60%, atleast 70%, at least 80%, at least 90% of the extent as the RBS having asequence as set forth in SEQ ID NO: 5 for the same nucleic acid sequenceunder identical experimental and transcriptional conditions (i.e. in thesame cell, with the same promoter etc.).

The RBS linked to the polynucleotide sequence encoding lcy-B is onewhich brings about expression of a nucleic acid sequence to at least60%, at least 70%, at least 80%, at least 90% of the extent as the RBShaving a sequence as set forth in SEQ ID NO: 2 for the same nucleic acidsequence under identical experimental and transcriptional conditions(i.e. in the same cell, with the same promoter etc.).

The RBS linked to the polynucleotide sequence encoding crtW is one whichbrings about expression of a nucleic acid sequence to at least 60%, atleast 70%, at least 80%, at least 90% of the extent as the RBS having asequence as set forth in SEQ ID NO: 2 for the same nucleic acid sequenceunder identical experimental and transcriptional conditions (i.e. in thesame cell, with the same promoter etc.).

The RBS linked to the polynucleotide sequence encoding crtZ is one whichbrings about expression of a nucleic acid sequence to at least 60%, atleast 70%, at least 80%, at least 90% of the extent as the RBS having asequence as set forth in SEQ ID NO: 2 for the same nucleic acid sequenceunder identical experimental and transcriptional conditions (i.e. in thesame cell, with the same promoter etc.).

As mentioned herein above, the present inventors further contemplateexpressing DXS. Preferably, the RBS linked to the polynucleotidesequence encoding DXS is one which brings about expression of a nucleicacid sequence to at least 60%, at least 70%, at least 80%, at least 90%of the extent as the RBS having a sequence as set forth in SEQ ID NO: 6for the same nucleic acid sequence under identical experimental andtranscriptional conditions (i.e. in the same cell, with the samepromoter etc.).

Using the methods described herein to express astaxanthin, the presentinventors obtained bacterial cells (E. coli cells) comprising more than2 mg/g cell dry weight of astaxanthin, comprising more than 3 mg/g celldry weight of astaxanthin, comprising more than 4 mg/g cell dry weightof astaxanthin, comprising more than 5 mg/g cell dry weight ofastaxanthin, comprising more than 10 mg/g cell dry weight ofastaxanthin.

Transformed cells are cultured under effective conditions, which allowfor the expression of high amounts of the recombinant polypeptides.Effective culture conditions include, but are not limited to, effectivemedia, bioreactor, temperature, pH and oxygen conditions that permitprotein production. An effective medium refers to any medium in which acell is cultured to produce the recombinant polypeptides of the presentinvention. Such a medium typically includes an aqueous solution havingassimilable carbon, nitrogen and phosphate sources, and appropriatesalts, minerals, metals and other nutrients, such as vitamins. Cells ofthe present invention can be cultured in conventional fermentationbioreactors, shake flasks, test tubes, microtiter dishes and petriplates. Culturing can be carried out at a temperature, pH and oxygencontent appropriate for a recombinant cell. Such culturing conditionsare within the expertise of one of ordinary skill in the art.

Following a predetermined time in culture, recovery of the astaxanthinis effected and spectrophotometric analysis or analysis by HPLC can beperformed.

The phrase “recovering astaxanthin” used herein refers to collecting thewhole fermentation medium containing the astaxanthin and need not implyadditional steps of separation or purification.

For collecting carotenoids and/or astaxanthin from bacterial cells or aculture solution following culturing, for example, bacterial cells maybe separated from a culture solution by a centrifugation or the like andextracted therefrom by an appropriate organic solvent. Examples of suchan organic solvent include methanol, ethanol, isopropyl alcohol,acetone, methyl ethyl ketone, methyl isobutyl ketone, dichloromethane,chloroform, dimethyl formamide and dimethyl sulfoxide. Among themacetone is preferred. Further, separation and purification into higherpurity may be achieved by utilizing a liquid chromatography or the like.Liquid chromatography may be based on a separation principle of ionexchange, hydrophobic interaction, and molecular sieve, for example.Reverse-phase chromatography and normal-phase chromatography arepreferred. Alternatively, extraction from cells may be conducted bysupercritical fluid extraction.

Alternatively, after completion of culturing, bacterial cells may beseparated from the culture solution by way of centrifugal separation,decantation, or filtration, for example. The obtained bacterial cellsare added with water to be rendered a slurry having a convenientviscosity. In order to prevent decomposition of carotenoids such asastaxanthin, an appropriate additive may be added to the slurry.Examples of such an additive include, but are not limited to,antioxidants such as ascorbic acid. Thereafter, the prepared slurry ishomogenized with the use of a grinder using glass beads or zirconiabeads or high-pressure homogenizer, and dried for use later. A preferreddrying method is spry drying.

The bacterial cells may directly be added to feeds for farm-raised fishor the like.

Alternatively, they may be extracted from a polar solvent or the like asdescribe above before use. Cell bodies remaining after extraction ofcarotenoids such as astaxanthin and containing little pigments can beused as ideal supply sources of proteins and vitamins in poultryraising.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

Examples

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

General Materials and Methods

Strains, Media and Reagents:

The bacterial strain used for cloning and construct assembly was E. coliDH5α unless stated otherwise. For the purification of plasmid DNA, cellswere cultured in LB media supplemented with suitable antibiotics at 37°and DNA was purified form culture using standard kits (Qiagen, Germany).All primers were synthesized by Sigma Aldrich, Israel. Detaileddescription of primers can be found in Table 2, herein below.

TABLE 2 RBS construction RBSX 1^(st) forwardGAATTCGCGGCCGCACTAGTTAATAGAAATAAT TTTGTTTAACTTTA - SEQ ID NO: 10 RBS AGATGGTGATGATGCATTCCAAACCTCCTTAAAG TTAAACAAAAT - SEQ ID NO: 11 RBS BGATGGTGATGATGCATCTCAGTACCTCCTCATTT TGTTTAAAGTTAAACAAAAT - SEQ ID NO: 12RBS C GATGGTGATGATGCATTCTTGCCTCTTAACTT TAAAGTTAAACAAAAT - SEQ ID NO: 13RBS D GATGGTGATGATGCATCGCCGCCGGTCCTGCTTATAAAGTTAAACAAAAT - SEQ ID NO: 14 RBS E GATGGTGATGATGCATCTTCCCCCTGCGAATAAAGTTAAACAAAAT - SEQ ID NO: 15 RBS Z GATGGTGATGATGCATCAGTGTATGGTGTAAAGTTAAACAAAAT - SEQ ID NO: 16 Cap Cassette construction F-primer +GCTAGCGTTGATCGGGCACGTAAGAG - SEQ ID NO: NheI(linker site) 17V1.0 - R primer GGCCACATGTCTGCAGCGGCCGCGCTAGC - SEQ ID NO: 18V1.1 - R primer GCGCTCGAGCGGCCGCGCTAGCTTATTACG -- SEQ ID NO: 19Cap Overhang GCTAGCGTTGATCGGGCACGTAAGAG - SEQ ID NO: addition 20Pviv primers KS f NotI + PstIgcggccgcctgcagGGCGGTAATACGGTTATCCA - SEQ ID NO: 21 Ks T1-T2 RCTCTCGCTTAGTAGTTAGACGTCCGACGTT GGAGTCCACGTTCT - SEQ ID NO: 22 KS T1-T2AGAACGTGGACTCCAACGTCGGACGTCTA pENTR11 F ACTACTAAGCGAGAG - SEQ ID NO: 23T1-T2 pENTER R actagtgaattcGCAACGAACAGGTCACTATC - SEQ ID NO: 24 BarcodeBar RBS A F CTCGAG AAA GTCGAC CTGCAG GCAGGGCGGTAATACGGTTA - SEQ ID NO: 25 Bar RBS b FCTCGAGGAG GTCGAC CTGCAG GCAGGGC GGTAATACGGTTA - SEQ ID NO: 26Bar RBS C F CTCGAG TAT GTCGAC CTGCAG GCAGGGCGGTAATACGGTTA - SEQ ID NO: 27 Bar RBS D FCTCGAG CAC GTCGAC CTGCAG GCAGGGC GGTAATACGGTTA - SEQ ID NO: 28Bar RBS E F CTCGAG AGG GTCGAC CTGCAG GCAGGGCGGTAATACGGTTA - SEQ ID NO: 29 Bar RBS Z FCTCGAG GGT GTCGAC CTGCAG GCAGGGC GGTAATACGGTTA- SEQ ID NO: 30 pRBS RTTACTTGTACAGCTCGTCCATGC - SEQ ID NO: 31Primers for amplification of fluorescence protein from Tsien collectionHis- Dye Forward ATGCATCATCACCATCACCACGTGAGCAAGGGCGAGGAG - SEQ ID NO: 32 Dye-Cap-ReverseCTCTTACGTGCCCGATCAACGCTAGCTTAC TTGTACAGCTCGTCCATGC - SEQ ID NO: 33primers for eliminating PstI site in mcherry (352) G353A ForwardCCAGGACTCCTCCCTGCAAGACGGCGAGTT - SEQ ID NO: 34 G353A ReverseAACTCGCCGTCTTGCAGGGAGGAGTCCTGG - SEQ ID NO: 35Haematococcus pluvialis isopentenyl pyrophosphate (ipiHp1)His-ipiHp1 Forward ATGCATCATCACCATCACCACCTTCGTTCGTTGCTCAGAGG - SEQ ID NO: 36 ipiHp1 ReverseATCACATCAACGAAGCGTGA GCTAGCGTT GATCGGGCACGTAAGAG - SEQ ID NO: 37eliminating 314 PstI site ipiHp1 T315C CACAAGTTCCTACCACATCAGCCCGCAGGCC -Forward SEQ ID NO: 38 ipiHp1 T315C ReverseGGCCTGCGGGCTGATGTGGTAGGAACTTGTG - SEQ ID NO: 39 ipiHp1 C435T ForwardGTGGACGAACACCTGCTGTAGCCACCCTTT SEQ ID NO: 40 ipiHp1 C435T ReverseAAAGGGTGGCTACAGCAGGTGTTCGTCCAC SEQ ID NO: 41Pantoea agglomerans geranylgeranyl pyrophosphate synthase (CrtE)His-CrtE ATGCATCATCACCATCACCACTATCCGTT TATAAGGACAGCC SEQ ID NO: 42CrtE Reverse CTCTTACGTGCCCGATCAACGCTAGCTTAACTGACGGCAGCGAGT SEQ ID NO: 43Pantoea agglomerans prephytoene pyrophosphate synthase (crtB)His-CrtB Forward ATGCATCATCACCATCACCACAATAATCCGTCGTTACTCAA SEQ ID NO: 44 CrtB Reverse CTCTTACGTGCCCGATCAACGCTAGCTTATGCCGGTACTGCCGGGC SEQ ID NO: 45Pantoea agglomeransphytoene dehydrogenase (crtI) His-CrtI ForwardATGCATCATCACCATCACCACAAAC CAACTACGGTAATTGGT SEQ ID NO: 46 CrtI ReverseCTCTTACGTGCCCGATCAACGCTAGC TCATATCAGATCCTCCAGCA SEQ ID NO: 47primers for eliminating PstI site in CrtI (478, 889) CrtI(G480T) ForwardGCACCTCAACTGGCGAAACTTCAGGCATGGA SEQ ID NO: 48 CrtI(G480T) ReverseTCCATGCCTGAAGTTTCGCCAGTTGAGGTGC - SEQ ID NO: 49 CrtI ( G891T) ForwardCGGTTAAGCAGTCCAACAAACTTCAGACTAA - SEQ ID NO: 50 CrtI (G891T)ReverseTTAGTCTGAAGTTTGTTGGACTGCTTAACCG - SEQ ID NO: 51Solanum lycopersicum beta-lycopene cyclase (LCY-B) His-LcY-B ForwardATGCATCATCACCATCACCACGATACTTTGT TGAAAACCCC - SEQ ID NO: 52 LcY-B ReverseCTCTTACGTGCCCGATCAACGCTAGCTCATT CTTTATCCTGTAACAAATTG - SEQ ID NO: 53primers for eliminating PstI site in Lyc-B (278) Lyc-B (A282G)GCTGTGGTTGGTGGTGGCCCTGCGGGACTT - SEQ Forward ID NO: 54 Lyc-B (A282G)AAGTCCCGCAGGGCCACCACCAACCACAGC - SEQ Reverse ID NO: 55primers for eliminating NsiI site in Lyc-B (1313) LycB (A1317G)CAAGAAGGTTCTTTGATGCGTTCTTTGACT - SEQ Forward ID NO: 56 LycB (A1317G)AGTCAAAGAACGCATCAAAGAACCTTCTTG - SEQ Reverse ID NO: 57Nostoc sp beta-carotene ketolase (CrtW) His-crtWATGCATCATCACCATCACCACGTTCAGTGT CAACCATCATC - SEQ ID NO: 58 CrtW ReverseCTCTTACGTGCCCGATCAACGCTAGCTTAT AAAGATATTTTGTGAGCTTCAGG - SEQ ID NO: 59Erwinia uredovora beta-carotene hydroxylase (crtZ) His CrtZ forwardATGCATCATCACCATCACCACTTGT GGATTTGGAATGCCC - SEQ ID NO: 60 CrtZ ReverseCTCTTACGTGCCCGATCAACGCTAG CTTATTACTTCCCGGATGCGGG - SEQ ID NO: 61

PCR reactions were performed using Phusion polymerase (Finnzymes,Finland). Cloning procedures were designed using Clone managerprofessional suite (Scientific & Educational Software, State Line,US-PA). Restriction enzymes were purchased from New England BioLabs(Beverly, US-MA) unless stated otherwise. Ligation reactions wereperformed using T4 DNA ligase (Fermentas, Lithuania). Detaileddescription of all of the equipment, reagents, suppliers and relevantcatalogue numbers can be found in Table 3, herein below.

TABLE 3 LB BROTH Conda 1231 Standard culture M9 Kanamycin Sigma AldrichIsrael Standard culture K4378-5G Chloramphenicol Sigma Aldrich IsraelStandard culture C0378-5G Ampicillin Sigma Aldrich Israel Standardculture A0166-5G Oligonucleotides Sigma Aldrich Israel PCR PhusionFinnzymes F-530S PCR dNTP Set Fermentase R0181 PCR Zymoclean Gel DNAZymo Research D4001 PCR cleanup Recovery Kit ATP Sigma Aldrich IsraelPNK A2383-5G phosphorilation cloning T4 DNA Ligase Fermentase EL0014Ligation Cloning T4 Polynucleotide Kinase NEB M0201L Self ligationcloning Alkaline Phosphatase, Calf NEB M0290L Cloning Intestinal (CIP)EcoRI-HF NEB R3101L Cloning SpeI NEB R0133L Cloning NsiI NEB R0127LCloning NheI-HF NEB R3131L Cloning XhoI NEB R0146L Cloning SalI-HF NEBR3138L Cloning PstI-HF NEB R3140L Cloning PciI NEB R0655L CloningQIAprep Spin Miniprep 27104 Miniprep Kit (50)

“No-Background” Assembly: General Cloning Scheme:

A “No-Background” assembly strategy was developed with the aim tofacilitate the serial assembly of multiple DNA sequences into a singleconstruct. All DNA constructs were generated according to themethodology described below. “No-Background” assembly adheres to theprinciples described in “Idempotent Vector Design for Standard Assemblyof Biobrick” (worldwide webdspacedotmitdotedu/handle/1721.1/21168), alsoknown as the BioBrick standard. In addition to preserving the mainfeatures described in the BioBrick system, an additional feature wasadded which eliminates the need to screen and validate that intermediateconstructs were correctly assembled throughout a multi-step assemblyprocess.

The key feature of the method is the concatenation of a chloramphenicolresistance marker (Cm^(R)): a constitutive promoter followed by aChloramphenicol Acetyltransferase coding sequence, to each of the DNAsequences designated for assembly. The Cm^(R) cassette is paired to theDNA sequence using PCR overlap extension prior to the assembly process.When the target DNA sequence (now paired with the Cm^(R)) is assembledinto a vector using a standard restriction-ligation process, only clonesthat were properly assembled are able to form colonies on agar platessupplemented with Cm.

Since the resistance cassette is flanked by restriction sites (FIG. 1),it can be easily removed when preparing the vector for the next assemblycycle. In this manner, it is possible to perform multiple assemblyrounds while using a single resistance marker. A general scheme of the“No-Background” Assembly strategy is described in FIG. 1.

Construction of Chloramphenicol Resistance Cassette:

The resistance cassette contains a constitutive promoter and achloramphenicol acetyltransferase gene as the resistance marker. Thecassette was amplified via PCR using the pSB3C5 plasmid as a template(BioPart: BBa_P1004). Restriction sites were added so the resistancecassette is flanked by NheI site at the 5′ and XhoI and PciI sites inits 3′ (Table 1, herein above).

Pairing Resistance Cassette with a Target Sequence:

Each of the DNA sequences designated for assembly were joined with theCm resistance cassette using a standard assembly PCR reaction. Aninsulator sequence containing an NsiI site was added upstream to thetarget sequence while the sequence GCTAGCGTTGATCGGGCACGTAAGAG (SEQ IDNO: 1) was added downstream. The latter sequence contains a NheI(underlined) site and a homology region of 20 bp to the beginning of theCm resistance cassette (bold). The homology sequence enables overlapextension PCR between the target sequence and the cassette, effectivelyenabling to pair the sequence of interest with the resistance marker.The PCR reaction was conducted using a sequence specific forward primerand a generic reverse primer (Cm-R, Table 1). The resulting PCR product(i.e. the target sequence concatenated to the resistance cassette) wasgel purified and may be sub-cloned into a vector or digested directlywith suitable restriction enzymes.

Choosing a Compact Set of RBS Sequences to Span Expression Space:

To find a small set of RBS sequences that span a large fraction of theexpression space, the forward engineered RBS series experimentallyanalyzed in the work of Salis et al., Nature Biotechnology 27, 946-950(2009) was used. First, the expected translation rate of each of the RBSsequences attached to various genes was computationally calculated[Salis Lab: The Ribosome Binding Site Calculator. atsalisdotpsudotedu/software/]. RBS sequences were chosen whose strengthseems to be the least affected by the downstream sequence. From thislimited set 5 RBS sequences were picked which spanned the largestexpression space experimentally [Salis et al., Nature Biotechnology 27,946-950 (2009)]. These RBS sequences were:

(SEQ ID NO: 2) #8 (RBS-A): AGGAGGTTTGGA (SEQ ID NO: 3) #1 (RBS-B):AACAAAATGAGGAGGTACTGAG (SEQ ID NO: 4) #17 (RBS-C): AAGTTAAGAGGCAAGA(SEQ ID NO: 5) #27 (RBS-D): TTCGCAGGGGGAAG (SEQ ID NO: 6) #20 (RBS-E):TAAGCAGGACCGGCGGCG (SEQ ID NO: 7) “Dead-RBS” (RBS-F): CACCATACACTG

Flanking “Insulator” Sequences:

Since the sequences flanking the RBS can affect expression levels,insulator sequences—a constant sequence of ˜20 bp located upstream anddownstream of each of the RBS were used. Such isolation sequences havebeen previously reported to be effective in reducing the effect offlanking sequences in the case of promoters. The upstream insulatorsequence was taken to be 19 base pairs, not natively found in E. coli:TAATAGAAATAATTTTGTTTAACTTTA (SEQ ID NO: 8) while the downstreaminsulator sequence was taken to be ATGCATCATCACCATCACCAC (SEQ ID NO: 9),a sequence coding for a 6His-tag.

RBS Modulation of a Target ORF:

In order to clone a target coding sequence for RBS modulation, it wasfirst amplified via PCR and paired to Cm resistance marker as describedabove. Once the target gene was paired to the resistance cassette, theproduct was ligated into a linearized BlueScript KS+ plasmid. The targetORF was then excised from the plasmid using either NsiI and PciI(non-barcoded assembly), or NsiI and XhoI. The resulting fragment,containing the target sequence and the resistance marker was thenassembled upon a RBS backbone vector (RBS backbone) containing a RBSsequence upstream to the insertion site. The resulting constructcontains the desired RBS followed by the target ORF and the resistancemarker, as described in FIG. 2.

pNiv—the Backbone Plasmid:

The backbone plasmid was constructed using Bluescript Ks+ as a base. TheLacZ gene was eliminated and the original multiple cloning site wasswapped with a new site that contains EcoRI, SpeI and PciI restrictionsites (FIG. 3). In order to minimize leaky gene expression throughoutthe assembly the process, the strong RRNB terminator (amplified by PCRusing pENTER11-Gateway as a template) was inserted upstream of the newcloning site. All DNA sequence modifications were accomplished by usingthe PCR overhang extension method.

pNiv:RBS-A-YFP to pNiv:RBS-F-YFP—Plasmid Set:

Each of the six core RBS sequences and the flanking insulator sequenceswere purchased as synthesized oligodeoxynucleotides. Each of the coreRBS sequences was flanked with the up- and downstream insulationsequences and fused to a YFP reporter gene in an assembly PCR reaction(see Table 1). The resulting six RBS-YFP reaction products (namely,RBS-A to RBS-F), were restricted and ligated into the backbone plasmidpNiv to yield the six designated plasmids—pNiv:[RBS-A to RBS-F]-YFP—seeFIG. 4.

Expression Plasmids:

The DNA assembly process was conducted on the pNiv backbone plasmidswhich contain no designated promoter. Once the assembly process wascompleted, the resulting product was sub-cloned into an expressionplasmid—pSB4K5:Ptac. This plasmid was derived from pSB4K5, a BioBrickstandard vector with low copy pSC101 replication origin (BioPart:BBa_I50042) and kanamycin antibiotic resistance marker (BioPart:BBa_P1003). LacIq Brick (BioPart: BBa_C0012) and a Tac promoter(BioPart: BBa_K301000) were assembled on pSB4K5 upstream to the multiplecloning site using standard assembly methods to yield pSB4K5:Ptac (FIG.5).

Experimental Measurements of RBS Expression Modulation Using FlowCytometry:

In order to quantify the effect of the RBS sequence on the expressionlevel in-vivo, a YFP reporter gene was placed upstream to each of theRBS sequence on a pSB4K5:Ptac plasmid using the cloning strategiesdescribed previously. E. coli MG1655 cells were transformed with thepSB4K5:Ptac-[RBS-A to RBS-F]-YFP plasmids and incubated at 37° C. inminimal media supplemented with 0.2% glucose until mid exponential phase(OD=˜0.3). Fluorescence was quantified using BD LSR II Flow Cytometer. Ablue laser (488 nm) and a 530±30 nm emission filter were used to measureYFP fluorescence and a yellow laser (560 nm) and a 610±20 nm emissionfilter were used to measure mCherry fluorescence. ˜100,000 cells wererecorded in each experiment (FIGS. 6A-D). Correlations between predictedRBS strength to experimental measurements are illustrated in FIG. 7.

pNiv-RBS Mixture Preparation:

Each of the six pNiv-RBS-YFP plasmids was separately digested with NsiIand PciI, removing YFP from the backbone vector. Digestion products weretreated with Calf Intestinal Alkaline Phosphatase (CIP) and gelpurified. An equimolar mix of the six resulting linearized vectors, eachdiffering only in the RBS sequence upstream to the cloning site wasprepared. This vector mixture (pRBS mix) was used to perform one-tubecombinatorial assembly with any target (FIG. 8).

Combinatorial Assembly of RBS Mixture:

For any coding sequence of interest, the coding sequence was cloned asdescribed above. In order to combinatorially pair the coding sequencewith the RBS set, the coding sequence was sub-cloned into the linearizedpRBS vector mixture. This resulted in a mixture of ligation products:all containing the same coding sequence but with a variety of RBS (RBS-Ato RBS-F) sequences upstream (FIG. 8).

Assembling a RBS-modulated synthetic operon: The resulting library,containing a mixture of constructs, all with an identical codingsequence but with a variety of RBS sequences upstream (RBS-A to RBS-F)can be used either as a vector or as an insert. First, by restrictingthe mixture using NheI and PciI, the resistance cassette is removed andthe plasmid library can be used as a vector into which more RBSmodulated coding sequences are assembled. Alternatively, by digestingthe mixture with SpeI and PciI, it is possible to excise the codingsequence (along with the upstream RBS) and use it as an insert forfurther assembly rounds.

In order to assemble a library of operons—where each variant containsthe same combination of genes but with a different combination of RBS,the RBS modulated mixture for each of the desired genes was constructedas described above. Iterative assembly steps were then performed, whereat each step an additional RBS modulated coding sequence was added alongthe operon. At every step, the product of the previous round wasdigested with NheI and PciI as shown in FIG. 9, removing the Cmresistance cassette. The reaction product was treated with CIP and gelpurified. The purified product, a linearized vector without Cmresistance, serves as a vector in the next assembly step.

To assemble an additional RBS modulated coding sequence to the operon,the RBS modulated mixture of the designated insert was digested usingSpeI and PciI, resulting in a DNA fragment which contains a mixture ofRBS sequences upstream to the coding sequence and the resistancecassette. This fragment was ligated into the operons library (alreadyharboring the first RBS modulated coding sequences). This assemblyprocess results in a combinatorial mixture RBS modulated codingsequences, where each variant has a distinct RBS composition upstream tothe coding sequences. The library was transformed into E. coli DH5α andplated on LB agar plates supplemented with Cm. The resistance cassettewhich was paired to the last incorporated gene ensured that onlyconstructs which contain the newly added RBS modulated sequence willcontinue for further assembly rounds.

Plasmid DNA from the newly constructed operon library was recovered fromthe plate by scraping the colonies directly from the plate andextracting the plasmids encoding for the operons library. Therefore, byrepeating this process for N rounds, where in each round an additionalRBS modulated coding sequence was added to the combinatorial operonlibrary, the present inventors sequentially assembled a combinatorialmixture of plasmids containing the same N coding sequences in apre-defined order and driven by a varying combination of the six RBS.

Barcoded RBS Mixture—Approach:

Since the operons library of RBS modulated coding sequences is built ina combinatorial manner, it is required to sequence all of the RBSsequences across the entire operon in order of determine the RBScomposition of a specific clone. To facilitate this process, andeliminate the need to sequence all of RBS spread across an operon, thepresent inventors assigned a 3 base pair barcode sequence to each RBS.These barcodes enable them to easily determine the complete RBScomposition of each clone using a single sequencing reaction at the 3′end of stacked barcodes as described in FIG. 10.

The barcodes are computed as follows: each RBS (A-F) was assigned anumber (1-6) which was then encoded in a 2-letter DNA code. A given2-letter code word (b₁b₂=AT, for example) maps to a particular number bythe formula b₁+4*b₂, where b₁ and b₂ are numeric values assigned to thebases of the code word (see table below). For the present example, ATbecomes 0+4*2=8.

TABLE 4 Base Numeric Value A 0 G 1 T 2 C 3

A third base was added as a check-base. This base was computed from theprevious two bases as follows: b3=(b1+5*b2) % 4, where ‘%’ representsthe modulus operator. This check-base allows detection of any singlebase mutation or sequencing error. Table 5 below provides the barcodevalues for each RBS. Note that, though only 6 RBS were used here, onecan potentially encode up to 16 RBS total using a 3 bp scheme. Theapproach is also scalable to much higher library sizes with alogarithmically scaled increase in code length.

TABLE 5 Code [with Check-Base] Numeric Value RBS-A AAA 0 RBS-B GAG 1RBS-C TAT 2 RBS-D CAC 3 RBS-E AGG 4 RBS-F GGT 5Addition of Barcodes to the pNiv-RBS Set:

Each of the six pNiv plasmid containing an RBS-YFP insert (RBS-A toRBS-F) served as template for a PCR reaction in which barcode bases andrestriction sites were added using designated primers (Table 2). XhoIrestriction site was added upstream of the barcode area while SalI andPstI sites were added downstream. Each of the six pNiv-RBS-YFP-barcodedplasmids was separately digested with NsiI and XhoI, removing the YFPcoding sequence. An equimolar mixture of the six resulting linearizedvectors (namely, pRBS-Barcode mixture) was prepared as described hereinabove.

Single Tube Combinatorial Assembly Using Barcoded RBS Mixture:

The use of the barcoded RBS plasmid set relies on the same logic asdescribed above except a few technical changes resulting from thedifferent use of restriction enzymes. The target coding sequence isfirst cloned as described above and digested using NsiI and XhoI. Theinsert is ligated with the pRBS-Barcode mixture and transformed in DH5αcells. A schematic description of the process is described below in FIG.11.

For simplicity the assembly process shown in FIG. 11 contains only twocoding sequences assembled into an operon, each with a specific RBS. Theprocess can be iteratively extended by additional assembly cycles.Moreover, a combinatorial RBS mixture can be used instead of specificones.

Subcloning into an Expression Plasmid:

After the assembly process was completed, the resulting operon wassub-cloned into an expression vector containing a designated promoter.This was obtained by using the designated restriction sites flanking thefinal operon. Moreover, since the expression plasmid had a resistancemarker differing from the Cm marker paired to the operon, whileselecting with both antibiotics, only positive colonies can grown whileclones transformed with either the self ligated expression plasmid orthe library donor plasmid could not.

RGB—Tricolor Reporter System

Bacterial Strains and Growth Conditions:

The bacterial strain used for the cloning and construct assembly processwas E. coli DH5α. For fluorescence measurements plasmids weretransformed into E. coli K12 MG1655 which were grown in minimal mediasupplemented with 0.2% glucose and chloramphenicol (34 ug/ml) at 37degrees.

Genes:

mYFP, mCFP and mCherry were amplified by PCR from the following plasmidspRSETB-YFP, pRSETB-CFP and pRSETB-mcherry. PstI restriction site onmCherry gene was eliminated by introducing a single silent mutation (seeTable 1).

Assembly Process:

mYFP, mCherry and CFP were first paired with resistance cassette asexplained above. The operon was assembled using the barcoded RBS set asdescribed in section above. pRBS-mYFP-barcode1 was digested with NheI &XhoI restriction enzymes in order to use it as a vector, whilepRBS-mCherry-barcode2 was digested with SpeI & SalI in order to use itas an insert. These two restriction products were ligated resulting innew product pRBS-mYFP-RBS-mCherry-barcode2-barcode1. This product wasdigested as a vector with NheI & XhoI, while pRBS-mCFP-barcode3 wasdigested as insert with SpeI and SalI, the resulting products wereligated to assemble the following operon in pNiv plasmid:pRBS-mYFP-mcherry-mCFP-barcode3-barcode2-barcode1. Next, the operon wasdigested sub-cloned into an expression plasmid as described above.

Measurements of RGB Fluorescence Library

Automated Fluorescence Measurements:

Cells were grown in a 96 well plate containing M9+0.2% Glucose in anautomated robotic platform (Evoware II, Tecan). Every 15 minutes theplate was transferred by a robotic arm into a multi-well fluorimeter(Infinite M200-pro, Tecan). In each measurement OD was sampled at 600nm, mCherry was sampled by excitation at 587 nm and emission measurementat 620 nm and YFP was sampled by excitation at 520 nm and emissionmeasurement at 555 nm.

Data Analysis:

Raw data of OD and fluorescence was background corrected by subtractingwells containing medium with no cells.

Because of the large required dynamic range, it was not possible toanalyze wells with weak RBS at low bacteria concentrations. Therefore,the present inventors chose to work at mid to late exponential phase.Cells were analyzed around an OD600 value of 0.1 as measured by theplate reader after media subtraction, equivalent to OD600 of ˜0.2 withstandard 1 cm path length. For each measurement point, the activity wasdefined as the increase of fluorescence during a time window of one hourcentered at the measurement's time divided by the average OD measuredduring that time:

${A(T)} = \frac{{F( {T + \tau} )} - {F( {T - \tau} )}}{\int_{T - \tau}^{T + \tau}{{{OD}(t)}{dt}}}$

where A is the RBS activity, F is the fluorescence measurement and τ=30minutes. The result reflects the increase in fluorescence during onehour divided by the number of cells. Mean activity was calculated byaveraging over 5 measurements around OD 0.1 for each sample:

$\overset{\sim}{A} = \frac{\sum\limits_{i = 1}^{l = 5}{A( t_{i} )}}{5}$

All analysis steps were performed using custom Haskell software.

Fluorescence Microscopy of Bacterial Colonies:

Fluorescence images were taken using a Nikon ECLIPSE E800 microscopeequipped with a Nikon Intensilight (C-HGFIE) for illumination. Chromafilter cubes set was used to image fluorescence proteins: mCherry(excitation filter 530-560 nm, emission filter 590-650, 30 ms exposure),cyan fluorescent protein (mCFP) (excitation filter 426-446 nm, emissionfilter 460-500 nm, 60 ms exposure) and yellow fluorescent protein (mYFP)(excitation filter 490-510 nm, emission filter 520-550 nm, 800 msexposure). Images were captured with a camera and NIS-Elements BR3.22software. Different channels were overlaid to give the figures shown.

Translational Coupling:

The translation of sequential genes within a single operon waspreviously shown to be dependent on upstream genes, a phenomenon termedtranslational coupling. Specifically, the expression level of a gene ismodulated by the expression level of the gene preceding it.Translational coupling was observed for various operons in E. coli aswell as other prokaryotes. While translational coupling has been knownfor many years, it is only crudely quantified and its underlyingmechanism is under debate.

The RGB grid shown in FIG. 16D demonstrates translational coupling.YFP's fluorescence depends only on the strength of the RBS controllingit, while mCherry's fluorescence depends both on the RBS controlling itand on YFP's fluorescence. To further analyze this effect the presentinventors utilized clones where YFP was controlled by one of the six RBS(A-F) while mCherry was controlled by either a weak RBS (E), a moderatestrength RBS (C), or a strong RBS (A). The fluorescence of YFP andmCherry for each library variant was then measured, and plotted againsteach other. The grid obtained (FIG. 13) clearly demonstratestranslational coupling: The first gene (i.e. YFP) expression leveldepends only on the strength of the RBS controlling it; the stronger theRBS used, the greater is the YFP fluorescence measured. In contrast, thesecond gene (mCherry) expression level depends both on the RBScontrolling it, and on the RBS controlling the first gene. That is,clones that share the same RBS for mCherry show different mCherryfluorescence level depending on the upstream YFP expression level. Crossfluorescence was ruled out as the cause for such an effect: clonescontaining a single fluorescence protein (either YFP or mCherry) do notgive a signal at the reciprocal fluorescence channel even for highlevels of expression. The maximal translation enhancement of mCherry byYFP expression level in the present system was found to be ˜6 fold. Thedependency between YFP and mCherry levels shows a linear dependence inlog space (FIG. 13). Linear regression gives a slope of ˜⅓ (95%confidence intervals 0.26-0.36).

RBS Modulation of the Carotenoid Biosynthesis Pathway

Bacterial Strains and Growth Conditions:

The bacterial strain used for the cloning and construct assembly processwas E. coli DH5α. For carotenoids expression, transformed cells weregrown in LB media supplemented with chloramphenicol (34 ug/ml) at 37degrees.

Genes for the Astaxanthin Biosynthetic Pathway

For the astaxanthin synthesis the following genes were used:

-   -   Geranylgeranyl pyrophosphate synthase (crtE) from Pantoea        agglomerans—GenBank: AAA21260.1 (SEQ ID NO: 62).    -   Prephytoene pyrophosphate synthase (crtB) from Pantoea        agglomerans—GenBank: AAA21264.1 (SEQ ID NO: 63).    -   Phytoene dehydrogenase (crtI) from Pantoea agglomerans—GenBank:        AAA21263.1 (SEQ ID NO: 64).    -   Isopentenyl pyrophosphate (idi) from Haematococcus        pluvialis—GenBank: AAC32208.1 (SEQ ID NO: 65).    -   Beta-lycopene cyclase (lcy-B) from Solanum lycopersicum—GenBank:        ABR57232.1 (SEQ ID NO: 66).    -   Beta-carotene hydroxylase (crtZ) from Pantoea        ananatis—Swiss-Prot: P21688.1 (SEQ ID NO: 67).    -   Beta-carotene ketolase (crtW) from Nostoc sphaeroides—GenBank:        BAB74888.1 (SEQ ID NO: 68).    -   1-deoxyxylulose-5-phosphate synthase (dxs) from Escherichia        coli—Swiss-Prot: A7ZX72.1 (SEQ ID NO: 69).

CtrZ was synthesized by using assembly PCR, the primers for the assemblyPCR were calculated using Johnson Lab Oligo maker (34). The restrictionsites EcoRI, SpeI, NsiI, NheI, PstI and PciI were eliminated from thelisted genes by introducing silent mutations.

Assembly Process:

idi, crtE, crtB, crtI, lcy-B, crtW, crtZ and dxs were amplified by PCRand then paired with resistance cassette as explained above. RBS wasadded to each gene as described above. The library was assembled in aniterative process according to the order of the genes along thebiosynthetic pathway. The complete operon was subcloned into anexpression plasmid as described.

Carotenoid Analysis

Carotenoid Extraction:

E coli cells carrying a plasmid with the biosynthetic genes of thecarotenoid pathway were grown in suspension cultures in shake flaskscontaining 100 ml of LB medium. Cultivation was carried out in 37° C. 20ml samples were withdrawn from the culture after 48 hours and cells wereharvested by centrifugation. Cell pellet was washed with cold water andcarotenoids were extracted by vigorous shaking with acetone (20 ml).Insoluble components of the extract were removed by centrifugation(15,000 g) and supernatant was transferred into a glass round-bottomflask and was evaporated using a rotary evaporator. Dried extract wasre-solvated in 1.5 ml acetone and 50-ul samples were taken for HPLCanalysis.

Carotenoid Analysis by HPLC:

HPLC analysis was performed on Jasco platform with high pressure mixinginstalled with a Borwin software, P4987 pumps and a MD-915 photodiodearray detector. Samples were analyzed by injecting 50 ul on a YMC packODS-A column (250×4.6 mm, 5 um, 12 nm). Solvent A: 75% aqueous methanol,Solvent B: ethylacetate. Solvent flow rate of 0.6 ml/min was used withthe following gradient: 15-85% of B (0-24 min), 85% (24-30 min), 85-15%(30-34 min), 15% (34-745 40 min). The spectra of the eluted carotenoidswere recorded online with the photodiode array detector (300-900 nm).Carotenoid compounds were identified by co-chromatography with authenticstandard compounds and by analysis of their UV-Vis spectra. For thequantification of the carotenoid compounds the integrated peak areaswere compared to those of authentic standards. The concentration of thestandard solutions was determined spectrophotometrically (Jasco V-570instrument)(35). For additional identification, the peaks isolated byHPLC were collected and directly injected into a mass-spectrometer(Micromass Quattro Ultima tandem quadruple instrument equipped with aZ-spray ESI interface and Waters Masslynx v4.1 software). Thecorresponding masses were analyzed from obtained full-scan (ESI(+), m/z100-1000) mass spectra(36).

Photos:

Pictures of colonies appearing in FIGS. 16A-D were taken using abinocular microscope (WILD M8; Heerbrugg, Switzerland) under visiblelight.

Results

Six RBS sequences that had previously been found to span several ordersof magnitude of protein expression were selected (9,14) (FIG. 15B).First, the effect of different RBS sequences was quantified onexpression levels by placing each sequence upstream to an YFP reporterand measuring the fluorescence signal using flow cytometry (15). The sixRBS were labeled ‘A’ to ‘F’ in descending order by expression level. Asshown in FIG. 15C, a small set of RBS can span several orders ofmagnitude of protein expression levels. Next, the present inventorsasked whether it was possible to use this small set of RBS sequences toassemble a library that spans the expression space of several genessimultaneously. A library of operons was generated where each membercontains the same genes but under the translational regulation ofdifferent RBS sequences. To achieve this, the present inventorscombinatorially paired each of the genes of interest with a set of RBSsequences and assembled these RBS-gene constructs to generate a libraryof synthetic operons (15).

An augmented BioBrick (16) cloning strategy was developed to facilitatethe assembly process. Genetic parts were iteratively assembled using apositive-selection procedure that bypasses the need for time-consumingscreening steps (15). Briefly, a chloramphenicol (Cm) resistancecassette was joined to all the genetic parts that were to be assembled.In each step, an additional genetic part was inserted into the construct(FIG. 15D) while the resistance cassette enabled a direct selection forproperly assembled constructs. The vector was then “recycled” for thenext iteration by excising the resistance cassette (FIG. 15D). Thisstrategy bypasses intermediate screening steps and enables fast andefficient operon construction. The resulting library of operons was thentransformed into cells and screened for a desired phenotype. Inferenceof the RBS composition of a specific clone was performed by sequencing abarcode located at the 3′ UTR of the gene (as described in the Materialsand methods). The barcode was generated during the assembly process byiteratively concatenating a short identifying sequence onto the 3′ UTRof the operon. Each genetic variant in the library contains a distinctbarcode sequence from which the RBS composition of all the genes in theoperon can be inferred in a single sequencing reaction.

To test whether RBS combinatorics can span a multi-dimensionalexpression space, a tri-color reporter system was constructed. CFP, YFPand mCherry were each randomly paired with three representatives of thepresent RBS set (RBS sequences ‘A’, ‘C’ and ‘E’) and assembled togetherinto an operon. The resulting operon library therefore contained 3³=27genetic variants, where each member contains the three genes in the sameorder but under the regulation of different RBS sequences (FIG. 16A).Upon transformation, colonies display distinct color patterns (FIG.16B), resulting from differential expression of the fluorescentreporters. The observed color space indicates that the combinatorialassembly of RBS sequences can significantly modulate expression level ofmultiple genes within the operon (FIG. 16C). The present inventorsverified that the different colors observed were attributed to differentcombinations of RBS sequences by sequencing sample clones andquantifying their fluorescence levels (15). In addition, the presentinventors measured the fluorescence levels of an operon consisting ofYFP and mCherry and found a grid of nine clusters as shown in FIG. 16D.Each cluster contains clones which have identical RBS. Moreover, thespread of the clusters demonstrates that RBS modulation spans ˜100 foldin each dimension of the expression space. Notably, the expression levelof YFP is dependent only on the RBS sequence regulating it. All colonieswith the same RBS upstream to YFP (located first in this operon) arealigned vertically and show a similar level of protein expression.However, mCherry fluorescence (located downstream to YFP) depends bothon its RBS and on the expression level of the upstream gene (FIG. 16D).Coupling over more than two orders of magnitude with a power lawexponent of ˜⅓ was found, showing an expression increases by about 2fold for every 10 fold increase in the expression of the upstream gene(FIG. 16D). The dependency is not due to cross fluorescence (15), but israther a manifestation of translational coupling (17) between adjacentgenes in the operon.

The present tri-color reporter system demonstrates that a combinatorialassembly of RBS sequences can span a large fraction of the expressionspace. The present inventors asked what the effect of such expressionmodulation had on the operation of a metabolic pathway. To address thisquestion, seven genes which compose the carotenoid biosynthesis pathwaywere cloned into E. coli. The end product of this exogenous metabolicpathway is astaxanthin, a high value xanthophylls (19) known for itspotent antioxidant properties. To explore the effect of combinatorialRBS modulation on the biosynthesis of astaxanthin in E. coli, each ofthe genes of the carotenoid pathway was randomly paired with the RBS setand assembled into a synthetic operon (FIG. 17A). The resulting librarycontains 6⁷ genetic variants in a single test tube. As shown in FIG.17B, the transformed E. coli colonies display a large variety of colorsand intensities. The color pattern of each colony is attributed todifferential accumulation of distinct carotenoid intermediates, eachhaving a unique color.

The RBS composition of sampled clones was determined by sequencing andthe carotenoid profile was analyzed using HPLC (15). FIG. 17C shows thatclones differing in their RBS composition exhibit diverse carotenoidprofiles. Some clones accumulate mainly a single product while othersproduced significant levels of a variety of carotenoids. Using thepresent combinatorial RBS approach, it was possible to increaseastaxanthin productivity to 2.6 mg/g of cell dry weight, about twice asmuch as previously produced in E. coli. Moreover, incorporating dxs—agene feeding into the carotenoid pathway—into the operon underRBS-modulation increased astaxanthin production to 5.8 mg/g of cell dryweight. This represents a 4-fold increase in astaxanthin production overthe best previously reported results.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

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What is claimed is:
 1. A method of generating astaxanthin comprisingexpressing polynucleotides encoding enzymes of an astaxanthin pathway,the polynucleotides comprising: (i) a polynucleotide which encodesPhytoene dehydrogenase (crtI) and a first transcriptional regulatorysequence; (ii) a polynucleotide which encodes Beta-lycopene cyclase(lcy-B) and a second transcriptional regulatory sequence; (iii) apolynucleotide which encodes Beta-carotene ketolase (crtW) and a thirdtranscriptional regulatory sequence; and wherein said first, second andthird regulatory sequence are selected such that the expression of saidlcy-B and said crtW is greater than a level of expression of said crtI.2. The method of claim 1, wherein each of said first regulatorysequence, said second regulatory sequence and said third regulatorysequence is a ribosome binding site (RBS).
 3. The method of claim 1,wherein said regulatory sequences are selected such that the expressionof said lcy-B and said crtW is at least five times greater than a levelof expression of said crtI.
 4. The method of claim 1, wherein saidregulatory sequences are selected such that the expression of said lcy-Band said crtW is at least ten times greater than a level of expressionof said crtI.
 5. The method of claim 1, further comprising introducinginto the cell a polynucleotide encoding a deoxyxylulose-5-phosphatesynthase (DXS).
 6. The method of claim 1, wherein said expressing iseffected in a bacterial cell.
 7. The method of claim 2, wherein each ofsaid RBS is flanked by a spacer sequence.
 8. The method of claim 1,wherein each of said polynucleotides are comprised on a singleexpression vector.
 9. The method of claim 1, further comprisingisolating the astaxanthin following said expressing.
 10. An isolatedpolynucleotide comprising: (i) a first RBS operatively linked to a firstenzyme coding sequence; (ii) a second RBS operatively linked to a secondenzyme coding sequence; and (iii) a third RBS operatively linked to athird enzyme coding sequence; wherein said second RBS is selected suchthat the level of expression of said second enzyme coding sequence isgreater than the level of expression of said first enzyme codingsequence; wherein said third RBS is selected such that the level ofexpression of said third enzyme coding sequence is greater than thelevel of expression of said second enzyme coding sequence; wherein saidfirst enzyme, said second enzyme and said third enzyme are non-identicalenzymes and each part of a biosynthesis pathway of an identical productof interest.
 11. The isolated polynucleotide of claim 10, wherein eachof said RBS is flanked by a spacer sequence.
 12. The isolatedpolynucleotide of claim 10, wherein said product of interest is aprotein.
 13. The isolated polynucleotide of claim 10, wherein saidproduct of interest is selected from the group consisting of a foodproduct, a pharmaceutical and a fuel.